Acetylgalactosamine (GalNAc)-siRNA Conjugates
Small interfering RNA (siRNA)-induced RNA interference (RNAi) responses have great potential to treat a wide variety of human diseases. A number of N-acetylgalactosamine (GalNAc)-siRNAs as RNAi therapeutics are currently in various stages of development. Creative Biolabs has accumulated extensive experience in offering our global clients with high-quality conjugation service of GalNAc-siRNA. Chemical modifications can be introduced to enhance the stability of GalNAc-siRNA.
N-Acetylgalactosamine Overview
N-acetylglucosamine is defined as a derivative of galactosamine, which can be synthesized through various chemical processes involving the protection and modification of functional groups to produce diazo derivatives, such as Ac4 GalDiaz. It is a stable compound used for the synthesis of bioorthogonal polysaccharides. Galactosamine is a model hepatotoxin that induces hepatitis characterized by neutrophil infiltration and kills animals through fulminant liver failure.
What is GalNAc-siRNA Conjugate?
The RNAi response induced by siRNA has great potential in the treatment of various human diseases, from cancer to pandemic virus outbreaks to Parkinson's disease. However, before siRNA becomes a drug, they must overcome billions of years of evolutionary defense mechanisms designed to prevent extracellular invasion of RNA into the interior of cells. It is not surprising that people have invested a lot of energy in developing various delivery technologies. The most important of these is the development of N-acetylglucosamine (GalNAc) siRNA conjugates for delivery to the liver. Tris GalNAc binds to the highly expressed sialoglycan receptor in liver cells, leading to rapid endocytosis. Although the specific mechanism by which siRNA penetrates the inner lipid bilayer membrane is not yet clear, a sufficient amount of siRNA entering the cytoplasm can induce a powerful targeted selective RNAi response in vivo.
Figure 1 (A) General illustration of conventional GalNAc-decorated oligonucleotides (siRNA) delivery mechanism and tracking strategy, (B) The mechanism of our DTM based tetra-antennary GalNAc-siRNA conjugate without incorporating extra fluorophores, (C) The chemical structure of a novel dithiomaleimides based tetra-antennary GalNAc. For simplicity, only trivalent GalNAc conjugates are indicated.1
How GalNAc-siRNA Conjugate Works?
- Key (GalNAc): GalNAc ligand attaches to siRNA molecule. It has a high affinity for specific receptors on the surface of liver cells.
- ASGPR: The asialoglycoprotein receptor (ASGPRS) is a protein that is extensively and specifically expressed on the surface of liver cells (hepatocytes).
- Targeted Delivery: When GalNAc siRNA conjugates are administered, GalNAc ligands bind to ASGPR on the surface of liver cells. This binding triggers a process called receptor-mediated endocytosis, in which cells engulf the binding complex and bring it into the body.
- Gene Silencing: Once inside the cell, siRNA is released from the binding complex and enters the cytoplasm, where it can bind to RNA induced silencing complexes (RISC). RISC uses siRNA as a guide for finding and disrupting complementary messenger RNA (mRNA), effectively silencing the expression of specific genes.
Core Services at Creative Biolabs
GalNAc is a sugar molecule that can recognize and bind to the cell surface protein sialic acid glycoprotein receptor (ASGPR), which is highly expressed in liver cells. If multiple GalNAc units bind to form multivalent ligands, the binding affinity with the receptor will increase exponentially. Creative Biolabs offers the service of binding three GalNAc molecules to siRNA, which means that three GalNAc molecules aggregate and bind to one siRNA molecule.
-
Custom GalNAc-siRNA Conjugate Design & Synthesis
- SiRNA Synthesis
- Conjugation Optimization
- Custom GalNAc Service
- Combination of GalNAc and siRNA
- Purification Characterization of GalNAc-siRNA Conjugates
Our Services Workflow
Creative Biolabs has established amino modifier services that can be of value throughout your research and development process, from start to finish. Here is our workflow for delivering customized, efficient, and quality solutions to you:
-
Phase I
Customer Consultation
Our process begins with in-depth consultation. This includes discussing target genes, disease indications, existing data, and any specific design considerations. We provide expert guidance for siRNA sequence design, potential chemical modifications, and appropriate conjugation strategies to optimize the application of GalNAc siRNA constructs. This collaboration phase ensures consistency in goals and lays the foundation for the success of the project.
-
Phase II
Project Design
After consultation, our R&D experts have developed a detailed project plan. This involves:
- SiRNA sequence optimization: Using complex bioinformatics tools and our proprietary algorithms, we design efficient and specific siRNA sequences while minimizing off target effects.
- Chemical modification strategy: Recommend and design a customized set of chemical modifications (such as 2 '- O-methyl, 2' - deoxy-2 '- fluoride, thiophosphate bonds) to enhance the stability of nucleases, improve pharmacokinetic properties, optimize cellular uptake and endosome escape.
- GalNAc coupling design: Select the optimal trivalent GalNAc linker and determine the precise coupling site on the siRNA double strand to maximize ASGPR binding affinity and target specificity.
-
Phase III
Project Execution
Using advanced bio coupling techniques to covalently link GalNAc ligands to modified siRNA. This critical step is strictly controlled to ensure specific and effective connections. Conduct in vitro studies to evaluate cell uptake, target mRNA knockout efficiency, and off target effects in relevant cell lines. For in vivo studies, we use appropriate animal models to evaluate pharmacokinetics (PK), pharmacodynamics (PD), tissue distribution (with a focus on liver specificity), efficacy, and preliminary safety.
-
Phase IV
Delivery of Results
- Detailed report: A complete report on synthesis, purification, and characterization data (such as HPLC chromatograms, MS spectra).
- Biological activity data: Results from in vitro studies (such as qRT PCR for mRNA knockout, Western blot for protein reduction) and in vivo studies (such as PK/PD profiles, efficacy curves, toxicity assessments).
- Material delivery: High quality, purified GalNAc siRNA conjugates that can be used for further customer specific applications or preclinical development.
Our Advantages
- Chemical modifications can be introduced.
- GalNAc-siRNA with well-tolerated in toxicology study.
- GalNAc-siRNA with enhanced stabilization chemistry.
- GalNAc-siRNA with high therapeutic indices, efficacy, and duration of action.
Frequently Asked Questions
Q: What are the main advantages of GalNAc siRNA conjugates compared to other siRNA delivery methods?
A: The main advantages are high specificity and liver targeting. GalNAc has high affinity and specificity to the asialoglycoprotein receptor (ASGPR), which is highly expressed on liver cells, resulting in efficient and rapid, receptor-mediated internalization of siRNA into liver cells. This obviates the need to deliver complex LNPs to liver and therefore, dramatically improves the therapeutic index for liver centered diseases.
Q: What is the role of RNA induced silencing complex (RISC) in the action of GalNAc siRNA?
A: The delivered GalNAc siRNA double stranded bodies are released into the cytoplasm after cellular uptake and endosome escape, where they are loaded into the RISC. In RISC, the siRNA double strand is separated and the antisense (guiding) strand is retained. Then, the guide chain guides RISC to identify and bind the complementary target messenger RNA (mRNA) sequences, which in turn is cleaved and degraded. This leads to silencing the expression of the corresponding protein.
Q: Why is GalNAc superior to other liver targeted delivery systems (such as LNPs)?
A: GalNAc siRNA conjugates have three key advantages over LNPs.
Targeting accuracy: ASGPR is only expressed on liver cells, while LNPs are taken up by multiple cell types (such as Kupffer cells and macrophages), leading to increased off target effects.
Convenience of administration: GalNAc conjugates require lower doses (0.1-1mg/kg vs. 1-10 mg/kg for LNPs) and can be administered subcutaneously (vs. intravenous injection of LNPs), making home administration possible.
Safety: LNPs can lead to liver toxicity (due to lipid accumulation) and immune activation (due to TLR4), while approved GalNAc siRNA (such as Inclisiran) did not have any serious treatment-related adverse events in phase III trials
Q: What modifications are needed to prevent siRNA from activating immunity?
A: The main driving factors are as follows:
TLR7/8 recognition: unmodified siRNA with high GU sequence content activates TLR7/8 in immune cells. We avoid this situation by:
- Use 2 '- OMe or 2' - F modifications in the passenger chain (downgrade after RISC loading).
- Design siRNA with low GU content (<30%).
- Complement activation: PS connection can activate the complement system; We will limit PS to 2-3 connections on the 3 'end to minimize this risk as much as possible.
Customer Review
"We are a startup developing GalNAc-siRNA conjugates for HBV, without the internal GMP production capacity. Creative Biolabs has been our partner of choice as they have delivered the conjugates on schedule and within budget. Their analysis team shared all quality control data including SPR binding affinity and in vivo efficacy in NHP which were used in our IND submission. When the US Food and Drug Administration asked for additional stability data, they expedited the testing and sent us the results within a week to enable us to meet the deadline for the trial start. We expanded our engagement to include preclinical safety study afterwards, which we are also very happy with their work."
—— Dr. Elara Vance, Head of HBV Therapeutics Development
Connect with Us Anytime!
Owing to their high predicted therapeutic indices and duration of action, GalNAc-conjugated RNA therapeutics have the potential to have a major impact in a variety of patient populations with high unmet need. In terms of the extensive experience in RNAi research, Creative Biolabs is proud to offer our clients with custom GalNAc-siRNA conjugations service. For more detailed information, please feel free to contact us or directly send us an inquiry.
Reference
- Kong S, Gao X, Wang Q, et al. Two Birds with One Stone: A Novel Dithiomaleimide-Based GalNAc-siRNA Conjugate Enabling Good siRNA Delivery and Traceability. Molecules, 2023, 28(20): 7184. https://doi.org/10.3390/molecules28207184 (Distributed under Open Access license CC BY 4.0, without modification.)
