These authors have contributed equally to this work and share first authorship
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RNA interference has become increasingly used for genetic therapy following the rapid development of oligonucleotide drugs. Significant progress has been made in its delivery system and implementation in the treatment of target organs. After a brief introduction of RNA interference technology and siRNA, the efficiency and stability of GalNAc-siRNA conjugates are highlighted since several oligonucleotide drugs of GalNAc have been approved for clinical use in recent years. The structure and features of GalNAc-siRNA conjugates are studied and the clinical efficiency and limitations of oligonucleotide-based drugs are summarized and investigated. Furthermore, another delivery system, lipid nanoparticles, that confer many advantages, is concluded, includ-ing stability and mass production, compared with GalNAc-siRNA conjugates. Importantly, developing new approaches for the use of oligonucleotide drugs brings hope to genetic therapy.
RNA interference (RNAi) is a natural defense mechanism widely present in organisms to protect against exogenous gene invasion (
N-acetylgalactosamine (GalNAc) conjugated to siRNA is considered as a promising solution of siRNA delivery system. The GalNAc GalNAc is targeting ligand binds highly selectively to asialoglycoprotein (ASGPR), which is abundantly expressed on hepatocyte cell, resulting in rapid endocytosis (
In this manuscript, several critical features are included. The structure and mechanism of action are priorly mentioned, following by the chemical modification, clinical and preclinical advances as well as challenges and limitations of GalNAc-siRNA conjugates. Finally, the features of GalNAc-siRNA conjugates are concluded by comparing GalNAc-siRNA conjugates to lipid nanoparticles (LNPs) to highlight the safety and efficiency of GalNAc-siRNA conjugates.
Despite its potential advantages, siRNA is beset with difficulties in clinical application (
Common nonviral vectors based on RNAi include LNPs, cationic polymers that represent polymer-based delivery systems and siRNA-carbohydrate bioconjugates such as GalNAc-siRNA conjugates (
Twenty years after RNAi discovery, siRNA therapy has begun to be implemented in clinical practice. In recent years, unprecedented progress has been achieved in developing various delivery technologies. One giant leap in delivery technology is the GalNAc-siRNA conjugate, which specifically delivers to and targets the liver. In November 2019, GIVLAARI® (givosiran), as the first GalNAc siRNA drug, was approved to be marketed in the United States for the treatment of acute hepatic porphyria, followed by inclisiran and lumasiran (
Marketed GalNAc-conjugated RNA drugs.
Brand name | Generic name | Target | Molecular mechanism | Condition |
---|---|---|---|---|
Leqvio | Inclisiran | proprotein convertase subtilisin/kexin type 9 (PCSK9) | PCSK9 Expression Inhibitors | atherosclerosis, dyslipidemia, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia |
Oxlumo | Lumasiran | hydroxyacid oxidase 1 (HAO1) | HAO1 Expression Inhibitors | end-stage renal disease, hyperoxaluria, primary hyperoxaluria type 1 |
Givlaari | Givosiran | 5′-aminolevulinate synthase 1 (ALAS1) | ALAS1 Expression Inhibitors | porphyria, acute porphyria |
Amvuttra | Vutrisiran | transthyretin (TTR) | TTR (Mutant) Expression Inhibitors | amyloidosis, transthyretin-related amyloidosis |
The coupling of drugs with cell surface receptor ligands has been reported as a promising pathway for targeted drug delivery. These receptors are only expressed in particular cell types and are overexpressed in specific organs or tissues in certain diseases. GalNAc is an efficient nucleic acid therapeutic ligand with a high affinity for ASGPR. ASGPR, also called the Ashwell-Morell receptor, has been established to feature liver specificity and species specificity. It is chiefly expressed on the surface of liver parenchyma cells in the hepatic sinusoid space and exhibits calcium-dependent ligand binding (
Synthesis of GalNAc-siRNA conjugates. Through chemically synthesized conjugates, modified siRNAs with GalNAc enter the system by subcutaneous injection, which is the most effective way to help GalNAc-siRNA conjugates attach to ASGPR, the receptor, that is, chiefly expressed on the surface of liver parenchyma cells in the hepatic sinusoid space, thus enabling ASGPR-mediated cellular endocytosis to cause siRNA to enter the cell and perform its function.
The GalNAc trimer is mainly synthesized by D-(+)-galactosamine (I), 2-amino-2-hydroxymethyl-1,3-propanediol (II), and trans-4-hydroxy-L-proline methyl ester (III). Solid carrier IV plus tyrosine can be used as a radioactive marker to evaluate the
GalNAc-siRNA conjugate pathway. The GalNAc-siRNA conjugate binds to ASGPR receptors on the surface of hepatocytes and is then endocytosed into the cytoplasm to form endosomes. Due to the decrease in pH in endosomes, the siRNA GalNAc-ASGPR complex is decomposed, and less than 1% of free siRNA escapes to the cytoplasm to exert an RNAi effect. ASGPR will return to the surface of liver cells for recycling, while GalNAc will be degraded and excluded. The negative chain of siRNA, Ago2 protein and related enzymes together constitute RISC and guide RISC to bind to target mRNA to achieve target mRNA gene silencing.
Moreover, the chemical modification of siRNA is conducive to further application of siRNA in the clinic. Initially, the experimenters try to modify the phosphate skeleton, base, terminal groups and ribose according to the structure of siRNA. The results show that ribose modification is more advantageous to some extent. Among them, the chemical modification of ribose 2′-OH is the most important. It is widely acknowledged that oligonucleotide nuclease stability can be significantly refined by adjusting the 2′ position of the RNA. In addition, it has been shown that by further improving the siRNA chemically, such as through optimizing the position of ribose modification of 2′-deoxy-2′–fluoro (2′-F) and 2′-O-methyl (2′-OMe) on the two strands of double-stranded siRNA (
Schemes of siRNA chemical modification.
Furthermore, nonclinical studies on ultratherapeutic doses of GalNAc-siRNA have shown that some typical molecules (six enhanced stable chemical GalNAc-siRNAs) exhibit similar safety signals and histological findings in the liver, as well as the kidney and lymph nodes (
To date, three GalNAc-siRNA therapeutics, Leqvio® (inclisiran), GIVLAARI™ (givosiran) and Oxlumo™ (lumasiran), have been approved for commercial applications, with 13 GalNAc-siRNA products undergoing clinical trials (
As previously mentioned, GalNAc-siRNA conjugates are commonly used in liver diseases (
Givosiran is an FDA-approved therapeutic siRNA based on GalNAc-siRNA technology. As previously discussed, givosiran is indicated for AHP caused by a disorder of hepatic ALAS1 and acute intermittent porphyria (AIP), which can result in the accumulation of several components, including toxic metabolites such as porphyrin precursors, ALA and PBG, leading to multiorgan and multisystem injury, including nervous system injury, along with conditions such as chronic kidney disease, hepatocellular carcinoma and hypertension (
Inclisiran is another GalNAc-siRNA conjugate approved by the FDA that can bind to hepatically expressed PCSK9 proteins, representing ideal targets that affect systemic and regional lipid metabolism and plasma cholesterol level regulation (
Lumasiran targeting glycolate oxidase is a promising therapeutic agent shown to effectively lower oxalate levels in the liver, resulting in less deposition of calcium oxalate crystals in the kidneys and lower probability of kidney failure, systemic oxalosis and other systemic organ failure due to metabolic dysfunction (
Vutrisiran is a transthyretin-directed siRNA therapeutic for the treatment of amyloid transthyretin-mediated (ATTR) amyloidosis, including hATTR amyloidosis and wild-type ATTR (wtATTR) amyloidosis (
As the representative of GalNAc conjugates clinical usage, Fitusiran, developed by Alynlam and Sanofi Genzyme, is a synthetic siRNA targeting liver antithrombin to increase thrombin generation developed for the treatment of coagulation factor VIII deficiency (hemophilia A) and coagulation factor IX deficiency (hemophilia B) and is ongoing. In a phase 1 inhibitor cohort, monthly fitusiran lowered antithrombin levels from baseline, resulting in thrombin generation improvements, suggesting that monthly subcutaneous injections of fitusiran may lead to bleeding episode reduction and improved quality of life in participants with hemophilia A or B treated with inhibitors (
The phase three ATLAS trial design consists of major branches including 1) ATLAS-A/B (NCT03417245), assessing fitusiran or on-demand factor replacement therapy, and 2) ATLAS-INH (NCT03417102), assessing fitusiran or on-demand bypassing agent therapy (
Additionally, preclinical undergone experiments with published papers are listed, with comparisons on years, models, animals, etc. (
Although GalNAc conjugates possess better stability, other crucial factors, such as endosomal escape, hepatotoxicity from off-target effects, acidic subcellular compartments, and extensive clearance, affect GalNAc efficiency and sequence barriers (
When GalNAc conjugates are functional
To eliminate hepatotoxicity, several solutions have been found. Janas et al. provide compelling evidence that off-target effects play key roles in hepatotoxicity during GalNAc treatment in rats (
It is shown that the results of GalNAc-siRNA conjugate loading into RISC match the corresponding model of GalNAc-siRNA conjugate liberation from the acidic subcellular compartment (
Given the wide range of nuclease degradation, sufficient RNA packaging is crucial to ensure that modified RNA can properly access receptors. Additionally, inadequate oligonucleotides exhibit quick renal filtration and elimination, leading to less intracellular residues and a lower half-life, significantly limiting treatment efficacy (
For many siRNA targets, the use of animal models to predict clinical results is prohibited because of the differential sequences between preclinical species. Therefore, new preclinical approaches are necessary in the development of oligonucleotide therapeutics to allow evaluation of target cell delivery as well as RNA silencing efficacy in species-relevant systems (
However, it was found that after years of development, available siRNA drugs still cannot solve problems associated with endosomal escape barriers, off-target effects and extensive enzymatic and/or systemic clearance. The targeting precision needs to be further improved (
The LNP delivery system, also known as the second-generation delivery system of RNAi drugs, is mainly composed of four parts: polyethylene glycol-lipid conjugates (such as PEG-DMG), ionizable amino lipids (such as DLin-MC3-DMA), distearyl phosphatidylcholine (DSPC) and cholesterol (
Comparison of GalNAc- siRNA and LNP loaded siRNA.
GalNAc-siRNA | LNPs loaded siRNA ( |
|
---|---|---|
Benefits & Challenges | -can be injected subcutaneously, with only small chances of plasma siRNA degradation, rapid absorption, high uptake, and long half-life, ( |
-avoid siRNA degradation of and the stimulation of the immune system by siRNA-lack efficient nuclear penetration and sustainable transgene expression (limitations such as poor biodistribution and possible toxic discharge), immunogenic and pose safety concerns of liposome molecules and limited efficacy and biosafety since unconjugated liposomes cannot achieve targeted delivery ( |
Composition | Specific arrangement of nucleoside, |
polyethylene glycol-lipid conjugates (PEG-DMG), ionizable amino lipids (DLin-MC3-DMA), distearyl phosphatidylcholine (DSPC) and cholesterol |
Attachment | Triantennary GalNAc ligand | PEGylated surface |
Target | ASGPR on hepatocyte | Liver and Triple-negative breast cancer (TNBC) ( |
RNAi activity ( |
slower | faster |
RNAi action time ( |
longer | shorter |
Approved drugs | Inclisiran, Lumasiran, Givosiran, Vutrisiran etc. | Patisiran ( |
GalNAc-siRNA conjugates are transmitted by ASGPR-mediated endocytosis and accumulate in acidic cells. It is mentioned that high metabolic stability is the most vital factor for GalNAc-siRNA to achieve optimal activity
Many chronic or acute serious diseases are associated with the liver, such as liver cancer, hepatitis, hepatic hemangioma, etc., which remain among the leading causes that seriously affect the quality of human life and even lead to human death. The immense potential of GalNAc siRNA conjugates in the treatment of liver-related diseases involving gene expression provides a new strategy for this purpose. Through years of improvement, siRNA therapeutics have achieved superior advances in delivery accuracy and targeting precision, and both LNP siRNA and GalNAc siRNA delivery systems have enabled siRNA systemic delivery. However, GalNAc conjugate-based siRNA capable of direct targeted delivery offers the advantages of being safer and more efficient than the second-generation delivery system LNP siRNA. Additionally, the results of intravenous injection of GalNAc-siRNA highlighted the durability of RNAi, while subcutaneous injection increased target gene knockout and prolonged the duration of RNAi activity. Moreover, studies have shown that chemically stable siRNA persists in highly acidic subcellular compartments after administration, with chemical stability contributing to prolonging the activity of RNAi. The progress in siRNA design and chemical modification is the crucial step in improving the stability of siRNA metabolism when designing oligonucleotides with continuously increased efficacy and enhancing the half-life of the GalNAc conjugates. To date, with several approved drugs and ongoing clinical and preclinical trials, it is promising that with more contributions taken into account, more delicate designs and modifications can lead GalNAc conjugates to not only longer duration but also higher efficiency, reduced toxicity and tighter attachment. Challenges such as endosomal escape barriers, off-target effects, and a lack of safety profile remain hindrances to actualizing the full opportunity and potential of oligonucleotide drugs. Thankfully, massive research is underway to investigate these obstacles, including the development of hepatic and extrahepatic delivery platforms, paving the way for further design and development. It is believed that state-of-the-art siRNA technology will surely be a boon to clinical medicine.
Conceptualization, ZL, LZ, YL, and XJ; methodology, GL; data curation, YZ; writing—original draft preparation, LZ, YL, and ZT; writing—review and editing, ZL, LZ, YL XJ, GL, and ZT. All authors have read and agreed to the published version of the manuscript.
This work has been financed by the National Natural Science Foundation of China (No. 81870591), Key R&D and Promotion Projects in Henan Province (Nos. 212102310874 and 222102310013), Key Scientific Research Projects of Colleges and Universities in Henan Province (No. 23A310011), National College Students’ Innovation and Entrepreneurship Training Program (Nos. 202110475021, 202110475048 and 202110475037).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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