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Design strategy and production optimization of RNA targeted drugs
Design strategy and production optimization of RNA targeted drugs. Stable chemical modification and delivery systems are the key to nucleic acid drug design strategies. Safety and efficacy are the main challenges faced in the design and development of RNA-targeted drugs, as well as the main reasons for the failure of early drug development.
1. The main challenges of RNA targeted drug design and development
Based on the clinical development experience of early RNA-targeted drugs, it is necessary to avoid the cytotoxicity caused by drugs. Safety issues are the main reason for the failure of early RNA-targeted drug development.
A key challenge in the development of RNA targeted therapies is to avoid non-specific toxicity. There are four main sources of cytotoxicity:
The immunogenic response of the innate sensor in the cell to foreign double-stranded RNA (dsRNA);
The immunogenic and non-immunogenic toxic effects of the delivery system and excipients;
Unexpected physiological activities caused by off-target drugs;
The accumulation of drugs in non-target tissues affects their physiological activities in targeted tissues.
The innate immune response to exogenous dsRNA comes from the induction of intracellular PKr, toll-like receptor 3 (tLr3) and tLr7. With the development of technology, it can now be modified by a wide range of 2′-MOE. This problem is largely avoided.
Toxicity of delivery system and excipients
The toxic effects of chemicals in excipients have plagued the development of drugs based on nanoparticle delivery systems and may be the main reason for the dose-limiting toxicity of many related drug candidates. Clinical trials have shown that triggers may directly come from the components of the excipients or the metabolic decomposition of the excipients. The person will change over time.
In addition, when cytotoxicity does occur, difficulty in determining the exact toxic component may be another major challenge. At present, the key strategy in clinical research may be to limit excipients to a small amount of chemical components. These chemical components are individually verified to be low-toxic. The assembled nanoparticles need to be as uniform as possible, which is closely related to improving the drug treatment window and reducing toxicity. Sex.
At the same time, nanoparticle formulations may degrade over time and lead to increased toxicity. Continuous quality monitoring of test drugs is likely to benefit future trials. Finally, pretreatment with glucocorticoids and anti-allergic drugs has greatly reduced the infusion reaction of drugs based on nanoparticle delivery systems.
Toxicity of off-target effects
Although ASO and siRNA drugs can specifically target target genes, the silencing of non-target genes may also occur in the seed zone match between the active ingredient of the drug and the non-target mRNA.
This problem can be improved by using tools such as Blast or other tools to eliminate targets that significantly overlap with target genes to screen for targets against the human genome sequence. However, the only way to ensure safety is through extensive testing, and it is crucial to use primate models that have a large amount of genome sequence overlap with humans. Even after extensive testing, some off-target effects may be inevitable, and clinical developers are trying to avoid these problems by minimizing drug dosages or using new base modifications.
Accumulated toxicity in non-target tissues
After RNA-targeting drugs are systematically delivered into the body, they will accumulate in many non-drug-active tissues.
Nowadays, RNA-targeted drug developers alleviate these problems by selecting highly disease-selective genes as targets for silencing, and by selecting delivery systems and delivery routes that reduce accumulation in non-target tissues. Future improvements in tissue-specific targeting of RNA-targeted drugs may alleviate these limitations and promote the development of other indications.
Another major challenge in the development of RNA-targeted drugs is the pharmacokinetic problem. After systemic administration, it is necessary to overcome the degradation of serum ribonuclease in the systemic circulation, so that the RNA drug can cross the cell membrane of the target cell, and then complete the endosomal escape. In order to ensure that a sufficient number of RNA molecules play a pharmacological effect after entering the cell, this has a profound impact on the efficacy of the drug.
With the development of technology, on the one hand, some chemical modifications can greatly improve the metabolic stability and PK properties, thereby making the drug more efficient. Currently, all ASO drugs approved by the FDA have undergone a large number of chemical modifications, and these drugs do not require additional The excipients can be distributed to target cells like small molecule drugs to achieve curative effect;
On the other hand, RNA-targeted drugs can be encapsulated by specific materials or coupled with ligands, and delivered through delivery systems such as viral vectors, thereby enhancing the targeting and efficacy of the drugs. In addition, like all other classes of drugs, the targeting and safety of RNA therapy are dose-dependent. When higher or larger doses of RNA-targeted drugs are used, dose-limited off-target effects are usually observed.
Therefore, in addition to designing the optimal sequence and using appropriate chemical/natural modifications to avoid or reduce immunogenicity and off-target effects, determining the correct dosage to achieve therapeutic effects and safety is critical to the development of RNA drugs.
2. Stability chemical modification
For RNA targeted drugs, chemical modification (except for tissue targeting ligands) has two basic functions:
First, chemical modification can greatly improve the safety of drugs by weakening the immune response of the cell’s endogenous immunosensor to dsRNA.
Second, by enhancing the ability of RNA drugs to resist degradation by endogenous endonucleases and exonucleases, the efficacy of the drugs is greatly improved. For siRNA drugs, chemical modification can also enhance the selectivity of the antisense strand to RISC loading, increase sequence selectivity to reduce off-target RNAi activity, and change physical and chemical properties to enhance delivery capabilities.
So far, all RNA-targeting drugs approved by the FDA are chemically engineered RNA analogs, supporting the utility of chemical modification. Single-stranded oligonucleotides for specific chemical modification categories differ only in sequence, but they all have similar physicochemical properties, so they have common pharmacokinetics and biological properties.
However, each chemical category is different, even a slight modification between 2′-methoxyethyl (2′-MOE) and 2′-methoxy (2′-OMe) may lead to efficacy , Significant changes in pharmacokinetics. Therefore, it is very necessary to accurately define the chemical properties of RNA-targeted drugs.
The specific types of chemical modification and their effects are as follows:
2’ end ribose replacement strategy
Oligonucleotide at the 2’end of the ribose hydroxyl group (-OH) can be replaced by MOE, OMe, F and other substituents, used to reduce immunogenicity, increase resistance to nucleases, improve plasma stability, thereby prolonging the drug effect.
(1) 2′-Methoxyethyl (2′-MOE): It can increase the PK of the drug, extend the elimination half-life to 2-4 weeks, improve the binding ability and effectiveness of the targeted mRNA, and reduce cytotoxicity;
(2) 2′-Methoxy (2′-OMe): It can improve the PK and stability of the drug, moderately improve the efficacy and reduce the immunogenicity;
(3) 2′-Fluorine (2′-F): It can improve the binding ability of the drug and the targeted mRNA but cannot improve the stability and PK. It is more suitable for siRNA drugs with RISC mechanism, and its modified nucleotide metabolites exist The possibility of integrating host cell DNA or RNA can lead to degradation of part of the nuclear protein.
In practical applications, the modification of the 2’end of ribose is incompatible with the activity of RNase H, which means that they are usually used for sterically hindered ASO or the flanking sequence of Gapmer ASO (such as Mipomersen, Inotesen introduced 2 ‘-MOE modification).
In addition, 2′-MOE modifications are usually not included in siRNA design. The structural requirements of Ago2 limit the types of chemical modifications that can be used. 2′-MOE has proven to be very useful for ASO with RNase H activity, but does not support binding to Ago2. siRNA.
5’end nucleic acid base modification strategy
5′-Methylcytidine (methylcytidine) and 5′-Methyluridine (methyluridine) modification can improve the binding ability of the drug to the target mRNA and reduce the immunogenicity, but they are only used for heterocyclic modification. Pyrimidine methylation can increase the melting temperature of each modified oligonucleotide by about 0.5°C, and improve the binding ability and stability of the drug to the target mRNA. It is usually used in ASO drugs (such as the ASO drugs being developed by Ionis Pharmaceuticals). Some use).
Ribose backbone modification strategy
The phosphodiester bond of oligonucleotides can be replaced by phosphorothioate (PS) bonds, that is, a non-bridging oxygen atom of the internucleotide phosphate group is replaced by sulfur, which has been widely used in the development of RNA targeted drugs.
The PS ribose backbone is a very effective modification. It has the dual effects of nuclease resistance and promotion of binding to plasma proteins, thereby reducing renal clearance, increasing the circulation time of drugs in the body, improving the pharmacokinetics of drugs, and removing drugs. The half-life is increased to 1-3 days. The PS ribose backbone supports RNA-targeted drugs with multiple mechanisms of action, especially in the application of Gapmer ASO and GalNAc siRNAs.
PS ribose backbone modification is easy to tolerate in ASO design and does not destroy RNase H activity. It is used in most ASO drugs that have been marketed. After systemic administration, it can be absorbed by most cells and does not require targeting ligands. . In contrast, siRNAs containing PS modification at each linkage site are less active than equivalent phosphodiester (PO) siRNAs. Therefore, siRNAs containing PS modifications are usually only modified at the end (if approved for marketing) Patisran).
One disadvantage of increasing the use of PS modifications in RNA targeted drugs is that each PS modification introduces a stereocenter with two possible chiral orientations, so an oligonucleotide with n PS modifications is 2n racemic Body mixture.
These two orientations have significantly different pharmacokinetic and pharmacodynamic properties. Although the PS bond in the Sp orientation has better resistance to nuclease cleavage, they also tend to reduce the number of bases compared with the Rp orientation. The dissolution temperature of the side bases reduces the stability. Since molecular heterogeneity is often detrimental to its clinical development, future RNA-targeted drugs may benefit from the recently developed PS-modified oligonucleotide stereoselective synthesis technology. For example, Wave Life Sciences has developed a scalable The method is used to synthesize oligonucleotides with fixed stereochemistry on each PS chain, and oligonucleotide drugs with fixed stereochemistry are being developed for various indications.
Another disadvantage of PS modifications is that they reduce the binding affinity of oligonucleotides to their targets. This limitation can be compensated by combining other types of modifications. It is worth noting that PS modifications increase drug resistance to cellular nucleases. This will lead to retention of the drug tissue and long-lasting drug effects. In order to cope with adverse reactions such as toxicity due to long-term gene silencing, the binding of one or more PO bonds can regulate oligonucleosides by reducing the stability of their nucleases. The durability of the acid.
2’ end ribose modification and nucleotide bridging strategy
Nucleotide bridges (BNAs) are confined in a fixed conformation by bridging between the 2nd and 4th carbon atoms of nucleotides. The most commonly used bridging strategies are LNA, cEt, ENA. BNAs enhance the drug’s resistance to nucleases and affinity to target mRNA (in LNA, the melting temperature of each modified nucleotide increases by 3~8°C).
Although BNA modified nucleotides are not compatible with RNase h, BNA modifications can be incorporated into the flanking regions of Gapmer ASO or used in sterically hindered ASO to improve target binding. It is worth noting that LNA-modified RNA-targeted drugs have observed hepatotoxicity and nephrotoxicity in some clinics, which increase the risk of sequence-related, and follow-up needs to continue to pay attention.
Other grooming strategies
Morpholino oligonucleotides (PMO) are a class of powerful synthetic oligonucleotide analogues. They belong to the third generation of antisense oligonucleotides in the history of nucleic acid drug development. The electrically neutral morpholino structure of PMO makes it It has the characteristics of high binding affinity and strong stability against enzymatic hydrolysis.
So far, two PMO-modified drugs (Eteplirsen and Golodirsen) have been approved by the FDA for marketing. PMO has the characteristics of high stability and safety. Because it is neutral at physiological pH and does not support RNase H1 activity, they are mainly used for drugs with steric hindrance mechanism. One disadvantage of PMO modification is that its lack of ability to bind albumin leads to lower PK properties, which means that it can be quickly eliminated by renal excretion, resulting in lower efficacy, so the drug dose needs to be increased.
It is worth noting that the PMO backbone contains chiral centers, which means that PMO drugs must be a racemic mixture. Unlike the PS modification described above, the effect of defining PMO stereochemistry has not been studied so far.
3. Delivery system
Regardless of the chemical modification, the size, hydrophilicity, and charge of RNA-targeted drugs pose additional challenges to systemic circulation, tissue extravasation, cellular uptake, and endosomal escape.
Because nucleic acid drugs need to enter the cell and complete the escape of the endosome in order to exert pharmacological effects. In order to overcome obstacles such as cellular uptake of nucleic acid drugs and low endosomal escape efficiency, delivery systems are necessary to improve drug targeting and bioavailability.
Currently, delivery systems developed for RNA-targeted drugs mainly include lipid nanoparticles (LNPs), polymers, nucleic acid nanostructures, exosomes, etc. RNA-targeted drugs can also be covalently bound to specific ligands. From relatively small molecules (such as aptamers, GalNAc, etc.) to large molecules (such as peptides, antibodies, etc. Bioconjugation), the targeted delivery of ligands is expected to improve the targeting of specific types of cells.
Lipid Nanoparticles (LNPs)
Originally developed as a delivery system for siRNA drugs in the body, LNPs are a complex structure (~100 nm) and are also used to deliver large RNA molecules such as mRNA in the body.
The disadvantage of LNPs in clinical applications is that their delivery is mainly limited to the liver and reticuloendothelial system, because the sinus-shaped capillary epithelium of the tissue provides a large enough space to allow these relatively large nanoparticles to enter. In addition, the local delivery of LNPs It has been successfully used to deliver siRNA to the central nervous system. LNPs can be further functionalized by polypeptides, PEGs, or other ligands that confer cell specific targeting.
However, it is worth noting that the increased complexity of LNPs will complicate their manufacturing and may increase their toxicity. This is a major issue that may limit its clinical application. For example, siRNA drugs coated with LNPs (such as Patisiran) need to be pretreated with steroids and antihistamines before intravenous injection to eliminate unnecessary allergic reactions.
In addition, biodegradable ionized lipids may enter the preclinical development stage in the next 2 to 5 years, which is expected to greatly increase the tolerated dose of drugs.
It is the most widely used ligand for nucleic acid drugs targeting liver cells. At present, about one-third of RNA targeting drugs in clinical trials are combined with multivalent GalNAc ligands to target the asialoglycose on the surface of liver cells. Protein receptors (ASGPRs) have greatly improved the targeting and bioavailability of nucleic acid drugs.
The surface of hepatic parenchymal cells expresses trimeric ASGPRs at a high level. ASGPRs can specifically bind to GalNAc at neutral pH, and release GalNAc in an acidic environment (pH 5-6), and then the released ASGPRs can be recovered into the cell The surface is reused.
Therefore, the suitable physiological conditions of the liver, the unique characteristics of ASGPRs, the non-toxic nature of GalNAc ligands and the ease of coupling make it a near-ideal method for systemic nucleic acid drug delivery to hepatocytes.
In addition, GalNAc-conjugated oligonucleotides can be efficiently administered by SC (adipose tissue below the epidermis and dermis) injection. Subcutaneously injected drugs are released into the systemic circulation at a slower rate and can also enter the lymphatic system. This gives cell receptors more time to regulate absorption. At the same time, subcutaneous injections are faster and easier, reducing the burden of treatment.
More importantly, the current clinically low incidence of local adverse events related to subcutaneous administration of GalNAc-conjugated oligonucleotide drugs, and the additional safety and tolerability will obviously support fewer once-weekly administrations. This is a comparative advantage with LNPs delivery system.
Coupling of therapeutic oligonucleotides and nucleic acid aptamers has also been used to enhance targeted delivery of siRNA and ASO. Aptamers can be thought of as chemical antibodies and can be designed as small nanostructures (about 20nm), which means that extrahepatic delivery is possible.
At present, Jingze Biology is developing a nucleic acid carrier platform based on DNA tetrahedral framework for the delivery of oligonucleotides, small molecules and other drugs. A variety of other delivery systems are also being developed in the clinic, which are not listed in this article.
Specific chemical modification can improve drug safety, metabolic stability, targeting, binding affinity and silencing effect, and greatly expand the therapeutic window of drugs. Therefore, the absolute necessity of chemical modification frustrated the early clinical development of RNA-targeted drugs China has been fully proven.
Delivery systems are the main bottleneck restricting the development of global RNA-targeted drugs. Lipid nanoparticles (LNPs) and N-acetylgalactosamine (GalNAc) have been verified in nucleic acid drugs approved for marketing. Various other delivery systems The development of is also undergoing clinical verification.
(source:internet, reference only)