Introduction: In 2006, the Nobel Prize went to siRNA drugs, in 2018, the world's first siRNA drug was approved, and in 2020, the first mRNA vaccine was approved for marketing, etc. All of these events shed lights on the great potential of nucleic acid drugs, which, as the third largest type of drugs after small-molecule and antibody drugs, are developing rapidly around the world. Nucleic acid drugs have become the focus and hotspot of research and development for biopharmaceutical companies. The development of nucleic acid drugs is extremely fast and highly targeted, and it is expected to make breakthrough on the "undruggable" targets that traditional approach cannot solve. On the other hand, the R&D and process barriers of nucleic acid drugs are still high. For example, the mechanism of action of drugs on the immune system has not yet been fully understood; delivery and patent modification protection are also extremely complicated. Regardless these hurdles, the field of nucleic acid drugs has entered an unprecedented historical period of vigorous development.
Introduction of Oligonucleotide therapeutics
Oligonucleotide therapeutics is a general term for state-of-the-art, molecular-target agents that employ chemically synthesized oligonucleotides with a single-stranded DNA or RNA backbone with potential specificity. These agents can inhibit gene expression or impede protein function by binding to a specific sequence of a target gene or protein.
Therapeutic oligonucleotides act on different stages of pathological gene expression. Standard schematic illustration of oligonucleotide activities in pathogenesis or disease progression. Decoys bind to transcription factors of targeted DNA at the earliest phase. Subsequently, antisense RNA, small interfering RNA (siRNA), and microRNA (miRNA) act to target mRNAs. Then, aptamers directly inhibit proteins in the process of pathogenesis.
Therapeutic oligonucleotides act on different stages of pathological gene expression[1]
Classification of Oligonucleotide therapeutics
Representative oligonucleotide therapeutics include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNAs (miRNAs), aptamers, and decoys. Among them, ASOs and siRNAs have been developed further than the others. ASOs are short, single-stranded nucleic acids that are typically 15-30 bases in length. siRNAs have a well-defined structure consisting of short (usually 20-24 base pairs) RNA duplexes with two base overhangs in the 3′ region.
Classification of Oligonucleotide therapeutics[1]
Global Development Status of Oligonucleotide Drugs
In recent years, with the advancement of related research and technology, nucleic acid drugs have ushered into rapid development, and the number of nucleic acid drugs approved for the global market is increasing yearly. Up to now, a total of 17 nucleic acid drugs have been approved for marketing in the world, including 15 oligonucleotide drugs (3 have been withdrawn from the market) and 2 mRNA products. The 15 oligonucleotide drugs approved for marketing include 9 ASO drugs, 5 siRNA drugs and 1 nucleic acid aptamer.
Approved oligonucleotide drugs (as of June 2022)[2]
Compared with foreign countries, there are currently no approved oligonucleotide drugs in China, and domestic companies with oligonucleotide drug pipeline are mostly in the early or rising stages of development. Such leading domestic companies include Abogen, Ribo, Sirnaomics, Ractigen. Globally, oligonucleotide drugs indications are widely distributed, covering tumors, genetic diseases, cardiovascular diseases, metabolic and other disease. Tumors and genetic diseases are accounted for the majority of indications in the clinical pipeline.
Mechanism of Oligonucleotide therapeutics
RNA interference (RNAi)
RNAi is a highly conserved natural process present in most eukaryotic cells in which double-stranded (ds) RNA molecules silence the post-transcriptional expression of specific genes. siRNAs and microRNAs are small non-coding RNAs consisting major mediators of the RNAi process. siRNAs have been used as synthetic mediators specifically designed to silence the expression of target genes. In miRNA and siRNA regulated pathways, this is known as the RNA-induced silencing complex (RISC) and drives silencing of a target mRNA via degradation and/or transcriptional repression.
Mechanism of RNAi[3]
Antisense oligonucleotides (ASOs)
ASOs comprise a promising class of synthetic agents designed to modulate gene expression. Antisense oligonucleotides target various classes of nucleic acids inside the cell (pre-mRNA, mRNA, non-coding RNA). ASOs inhibit protein production mainly through stimulation of RNAase H activity, which in turn results in target mRNA degradation (ASO “Gapmers”)
Mechanism of ASOs[3]
Aptamers
Aptamers are single-stranded DNA or RNA molecules selected through a large oligonucleotide library, called SELEX, to bind a specific target with high selectivity and specificity. Common targets include small metal ion and organic molecules, proteins, viruses, bacteria and whole cells. Target recognition and binding involve three-dimensional, shape-dependent interactions as well as hydrophobic interactions. Here is a schematic illustration of the aptamer Pegaptanib inhibiting the action of the target protein VEGF-165 by binding to its receptor VEGFR.
Mechanism of Aptamers[3]
Advantage of Oligonucleotide therapeutics
❖ Highly specific
❖ High efficiency of knockdown of genes
❖ Produce longer lasting response
❖ Potential sensitivity to therapy can be measured
❖ Reduced R&D cycle time
❖ Abundance targets
Pros and cons comparison of oligonucleotide versus small molecule drugs[4]
Development Challenges and Strategies of Oligonucleotide Drugs
The production of oligonucleotide API uses solid-phase synthesis technology, which has high barriers in process development, process scale-up and quality control. The initial investment in the solid-phase synthesis equipment and clean environment for making oligonucleotide API is very large, and the production must meet the requirements of GMP. There are fewer domestic enterprises capable of producing oligonucleotide APIs, and the related industries are not yet perfect. With the increase in market demand, the timely supply of oligonucleotide drugs has become an important challenge for the success of product development and commercialization.
Nucleic acid drug synthesis & modification:
At present, the small nucleic acid platform of Medicilon Chemical Department can undertake one-stop drug discovery services for the modification and synthesis of nucleotide monomers; the synthesis of delivery systems; the synthesis of oligonucleotides, and oligonucleotide conjugates. The siRNA library that has been built in Medicilon not only has a rich monomer inventory, but also includes a huge building block library for monomer synthesis, which allows quickly completing the synthesis of various modified monomers. Further, Medicilon has a professional small nucleic acid drug R&D team that can provide efficient and fast R&D services; several siRNA and other oligonucleotide drug FTE projects have been completed or in good progress.
Nucleic acid drug CMC research:
The pharmaceutical challenges in the development of oligonucleotide drugs are mainly due to the fact that the raw materials and equipment used in the production process rely on large-scale production capacity to meet the commercialization need of the product. In the process of expanding production, many aspects need to be considered such as product quality, production speed and cost, and strong restrictions need to be complied with as well. Medicilon's CMC is now providing service for oligonucleotide drugs such as siRNA.
Nucleic acid drug Pharmacodynamics:
Pharmacodynamic challenges in oligonucleotide drug development include the followings: Insufficient targeting due to the low concentration of oligonucleotide drug at the target site leading to an increased doses administered; off-target toxicity effects caused by the binding of oligonucleotide drugs to non-target RNA and more.
Medicilon Case：Pharmacology evaluation
❖ Comparing different drug delivery methods (IV vs intra-tumor injection)
❖ Correlation analysis between nucleic acid drug efficacy, targeted mRNA/protein degradation and drug PK
Nucleic acid drug bioanalysis:
Medicilon Case：Compound A –siRNA plasma quantification (20 µL plasma)
Nucleic acid drug pharmacokinetics:
The pharmacokinetic (PK) challenge in the development of oligonucleotide drugs is considered the biggest for this type of drug. Because nucleic acid drug is easy to be degraded by plasma nucleases, its serum stability is poor, and quickly cleared out of the body by the kidneys, so the circulation time of oligonucleotide drugs in the body is short. In addition, after entering the cell, oligonucleotide drugs tend to accumulate in the nucleus and rather than entering the cytoplasm to play a role. Therefore, what must be considered in the development of oligonucleotide drugs is that how to keep drugs in the body for a long enough time, and accurately enter the targeted cells to exert therapeutic functions, thus avoiding accidental injury to normal cells to the greatest extent when injecting oligonucleotide drugs into patients.
Oligonucleotide drugs are unstable in the body, easily degraded by nucleases in circulation, and cleared through the kidneys with a short half-life. At the same time, exogenous nucleic acid molecules are immunogenic and easily trigger immune reactions in the human body. In addition, oligonucleotide drugs will not be effective if they cannot enter the cell through endocytosis. With technological breakthroughs, some problems have been better solved at present, among which chemical modification (such as: phosphate backbone, ribose, ribose five-membered ring modification, base, nucleotide modification, etc.) can avoid the degradation of nucleic acid drugs by nucleases and prolong the half-life. Efficient and safe delivery systems (such as cyclodextrin nanopolymers, lipid nanoparticles, conjugate delivery systems, acetylgalactosamine systems, etc.) can precisely target nucleic acid drugs to target cells and improve the efficiency of cell uptake, so that nucleic acid drugs can exert therapeutic functions.
Oligonucleotide drug treatments focus on the post-transcriptional level, and can achieve breakthroughs for special protein targets that are difficult to drug, and are expected to overcome diseases that have no drug treatment, including genetic diseases and other intractable diseases.
Barriers to Nucleic Acid Drug Delivery[5]
Medicilon Liver Biopsy Guided By B-ultrasound In Cynomolgus Monkeys Platform
The development of gene therapy and nucleic acid drugs has made the establishment of monkey models and related research a hot topic. Due to the high similarity of genetic, morphological, physiological and biochemical characteristics with humans, non-human primates, especially cynomolgus monkeys, are closest to humans in terms of evolution, and have outstanding advantages in model construction, disease mechanism research, and drug development. Many disease models have been established so far.
In the long-term dynamic experimental observation of the changes in the liver disease model of cynomolgus monkeys, due to the limitations of animal disease models and experimental objective conditions, researchers mostly obtain liver tissue pathological analysis and diagnosis of these disease models through blind puncture or surgical sampling, which not only causes great trauma to animals, complicated postoperative care, but also easily leads to various complications, which is not conducive to long-term observation of disease models.
The Medicilon Liver Biopsy Guided By B-ultrasound In Cynomolgus Monkeys Platform can avoid the large blood vessels and gallbladder to the greatest extent, and has the advantages of less trauma, safe and simple puncture operation, accurate positioning, and better postoperative recovery. Medicilon Liver Biopsy Guided By B-ultrasound In Cynomolgus Monkeys Platform can dynamically display the whole process of biopsy needle insertion and material collection in real time, which greatly improves the success rate of puncture and the accuracy of experimental results.
At the same time, it can be used for the preclinical PK evaluation of gene therapy drugs, which also promotes the improvement of experimental animal welfare, and provides accurate pathological basis for the dynamic monitoring and modeling progress of various liver disease models.
Medicilon DMPK department conducts Cynomolgus Monkey liver biopsy for PK/PD research
Market Prospects of Oligonucleotide Drugs
Oligonucleotide drugs have the advantages of high specificity, convenient design, short development cycle, a variety of targets, thus are becoming the focus of current research in the field of biomedicine. At the same time, the development of nucleic acid drug delivery platforms (conjugated delivery systems, nanoparticle carriers, etc.) will promote the development of delivery technology and multiple drug delivery routes that could be widely applied in drug discovery (such as: subcutaneous injection, intravenous injection, aerosol inhalation, intra-tumoral injection, etc.).
The continuous breakthroughs and innovations in the fields of application and technology of oligonucleotide drugs will help the development of oligonucleotide drugs. Market demand and market scale will continue to expand, and cover a wide range of indications, including tumors, rare diseases, viral diseases, kidney diseases, cardiovascular diseases, inflammatory diseases, metabolic diseases, etc. Therefore, oligonucleotide drugs could be beneficial to different patient populations with a variety of diseases. With the development of technology and the maturity of production, the oligonucleotide drug market will have a broader development space in the future.
References:
[1] Kazuki Takakura, et al. The Clinical Potential of Oligonucleotide Therapeutics against Pancreatic Cancer. Int J Mol Sci. 2019 Jul 6;20(13):3331. doi: 10.3390/ijms20133331.
[3] Ageliki Laina, et al. RNA Therapeutics in Cardiovascular Precision Medicine. Front Physiol. 2018 Jul 25;9:953. doi: 10.3389/fphys.2018.00953. eCollection 2018.
[4] Phuc Tran, et al. Delivery of Oligonucleotides: Efficiency with Lipid Conjugation and Clinical Outcome. Pharmaceutics. 2022 Feb 1;14(2):342. doi: 10.3390/pharmaceutics14020342.
[2] Seong Jun Jo, et al. Clinical Pharmacokinetics of Approved RNA Therapeutics. Int J Mol Sci. 2023 Jan 1;24(1):746. doi: 10.3390/ijms24010746.
[5] Xuyu Tan, etal. Nucleic acid-based drug delivery strategies. JControl Release. 2020 Jul 10;323:240-252.
[6] David Bumcrot, et al. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol. 2006 Dec;2(12):711-9. doi: 10.1038/nchembio839.
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