The core technologies of oligonucleotide drug development include sequence design, synthesis and modification, isolation and purification, and delivery techniques. The production of oligonucleotide raw materials adopts solid-phase synthesis technology, with high barriers to process development, scaling up, and quality control. The upfront investment includes equipment for solid-phase synthesis of oligonucleotide raw materials and a clean environment, which must meet GMP requirements. With increasing market demand, timely supply has become an important challenge for product development and commercialization.
Sequence design of oligonucleotide drugs
Small nucleic acid drugs recognize target mRNA based on the principle of complementary base pairing, unlike antibodies and small molecule drugs that require complex protein spatial structures for recognition. The design of small nucleic acid drugs only requires the target mRNA sequence, without the need for large-scale drug screening, so the current barriers to drug sequence design are relatively low. When designing small nucleic acid drugs, factors such as homology, immunogenicity, conservation, and off-target effects must be considered to obtain high specificity and potentially active high-quality drugs. Sequence design typically involves appropriate length, nucleotide content, and base sequence.
Synthesis and modification of oligonucleotide drugs
Small nucleic acids have poor stability and low specificity and require different chemical modifications to improve their stability and ability to be absorbed by tissues and cells. Methods for improving the drug-like properties of oligonucleotide drugs by modifying the phosphate backbone, sugar component, and base itself have been widely used and can enhance delivery efficacy. These different modification methods can greatly improve the pharmacokinetics, pharmacodynamics, and biodistribution of oligonucleotides.
Isolation and purification of oligonucleotide drugs
Due to the impact of multiple factors such as synthesis efficiency and raw material purity, various impurities can exist after the synthesis of oligonucleotide drugs. Common impurities include oligonucleotides with missing or added sequences, products with unremoved protecting groups, oligonucleotides with missing purine bases, and other degradation products. To purify these crude products after synthesis, ion exchange, hydrophobic, and reverse-phase chromatography methods are commonly used, and the purification method needs to be selected based on the characteristics of the base sequence. Ordinary DNA, RNA, or modified base sequences are generally purified using ion exchange resin, while those with 3′ or 5′ additions of PEG or other delivery systems (LNP liposomal nanoparticles) often require high-pressure column purification and separation. For the purification process of oligonucleotides, a balance needs to be achieved between product purity and yield. Therefore, effective purification and amplification strategies and fraction collection analysis are crucial for establishing a good process space during process development and scale-up.
CMC research of nucleic acid drugs
In the development of oligonucleotide drugs, pharmaceutical challenges include large-scale production and high requirements for analysis and quality control. Large-scale production requires high requirements for monomer raw materials, equipment, synthetic processes, and purification. In terms of analysis, oligonucleotides have a high degree of structural similarity to related substances and active ingredients, and therefore, multiple principles and methods are required for quality research and control.
Delivery technology of oligonucleotide drugs
Small nucleic acid drugs typically face two major challenges when entering cells: first, RNA is easily degraded by nucleases in plasma and tissue when exposed in the bloodstream, and second, negatively charged RNA is difficult to cross the cell membrane. Due to delivery system and immune issues, progress in clinical research has been slow. Breakthroughs in chemical modification and delivery system technology are critical to the development of nucleic acid drugs. Although chemical modification can improve the stability of nucleic acid drugs and reduce immunogenicity, these drugs must enter cells in order to be effective. Because nucleic acid drug molecules are large and usually carry a negative charge, the efficiency of cellular uptake and endosome escape is relatively low, therefore, help from a delivery system is needed. Currently, drug delivery systems are divided into virus carriers and non-virus carriers. Virus carriers are commonly used in gene therapy, but they are less frequently used in nucleic acid drugs due to their immunogenicity, tumorigenicity, and limited drug-carrying capacity. Non-virus carriers are more widely used, including polymers and lipids (such as liposomes or LNP). In addition, nucleic acid drugs can be combined with specific ligands to achieve targeted delivery to specific cells, such as GalNAc, peptides, and antibodies.
Common delivery systems of oligonucleotide drugs
Small nucleic acid drugs, especially RNAi drugs, must cross the cell membrane to act on mRNA molecules in the cytoplasm or nucleus. However, this delivery process is very difficult. Although chemical modifications can solve the problems of stability and immunogenicity, if small nucleic acid drugs cannot enter cells and achieve endocytosis, they will not be able to exert their pharmacological effects. This problem has been a bottleneck in the industrialization process of small nucleic acid drugs. With technological advances, delivery systems have become the key to solving various problems. Currently, delivery systems used in small nucleic acid drug research and development differ in structure, shape, size, chemical properties, and mechanisms of action. Common delivery systems include: 1) GalNAc ligand-modified short interference RNA (siRNA) conjugates; 2) lipid nanoparticles (LNPs); 3) peptides and antibodies, among others.
GalNAc Conjugate Delivery System
GalNAc (N-acetylgalactosamine) is a ligand for the asialoglycoprotein receptor (ASGPR), which is an endocytic receptor highly expressed on the surface of liver cells with high specificity and low expression in other cells. In the 1960s and 1970s, scientists discovered that lactose could bind to the CDR receptor on the liver surface and be internalized, so they used this characteristic to deliver proteins, peptides, small molecules, and other substances into cells. In 2008, Alnylam demonstrated that GalNAc-conjugated nucleic acid drugs could be successfully delivered to the liver. Alnylam covalently linked GalNAc units in a trivalent state to the 3′ end of the sense strand of siRNA, with three GalNAc molecules aggregating and conjugating to one siRNA molecule, ensuring high affinity between the ASGPR and GalNAc ligand and promoting optimal siRNA delivery to the liver. According to Alnylam’s research, GalNAc-siRNA conjugates can rapidly enter the liver after subcutaneous injection and rapidly decrease plasma concentration. After entering the liver, they are rapidly internalized by liver cells mediated by the ASGPR, accumulate in lysosomes, slowly release, and persistently load onto RISC, achieving long-lasting inhibitory effects. Following Alnylam’s first siRNA approval, the next four siRNA drugs were all based on the GalNAc delivery system. The GalNAc conjugate technique can be applied to ASO drugs and siRNA drugs and is the most effective nucleic acid drug delivery system to date but has limitations and can only target liver cells.
Lipid nanoparticles (LNP) are currently the mainstream carrier delivery method. Because they are easily absorbed by antigen-presenting cells, they are often used in vaccines. Currently, the three major mRNA vaccine giants Moderna, CureVac, and BioNTech all use LNP delivery technology for their COVID-19 vaccines. LNP is a spherical solid nanoparticle containing lipid components, including ionizable cationic lipids, neutral helper lipids, cholesterol, and PEG-modified lipids. siRNA is encapsulated in LNP for targeted tissue delivery. LNP protects siRNA from degradation by nucleases and delivers siRNA into cells to exert its action by fusing with the cell membrane.
Peptide Delivery Vectors
Peptides have the characteristics of small molecular weight, low immunogenicity, high specificity, and renal excretion, which have the advantages of both large and small molecules. As of now, peptides used for drug delivery are classified as cell-penetrating peptides (CPP), cell-targeting peptides (CTP), self-assembling peptides (SAP), and responsive peptides. Peptides can exhibit targeting functions in various application scenarios. By forming peptide-oligonucleotide conjugates (POC), they can transfer load molecules to target cells and tissues with specificity. Whether through covalent bonding or the formation of nanocomposites with load molecules, peptide delivery carriers have great mobility advantages, which can enter challenging tissues such as muscles, bone marrow, and the blood-brain barrier. Taking CPP as an example, CPP is a short peptide that can transport small molecule payloads across cell membranes. POC as a potential therapeutic drug has many important functions and can resist the action of intracellular enzymes existing in different cellular compartments due to its stability. In terms of covalent bonding methods, thiol-maleimide coupling is the preferred method, which is widely used in peptide chemistry and has been approved by regulatory authorities for various ADC drugs.
Trend of oligonucleotide drug development
With breakthroughs in the application and technology of oligonucleotide drugs, updated technology will drive the development of oligonucleotide drugs. Market demand will continue to expand, and oligonucleotide drugs have a wide range of applications, including tumors, rare diseases, viral diseases, kidney diseases, cardiovascular diseases, inflammatory diseases, metabolic diseases, and so on. Therefore, the potential target population for oligonucleotide drugs is enormous, and as technology and production mature, the oligonucleotide drug market will have a broader development space.
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