Tetra-antennary GalNac-RNA Delivery Service
Overview
The quest for highly effective, safe, and conveniently administered nucleic acid therapeutics culminates in targeted delivery. CD BioGlyco is proud to introduce the tetra-antennary GalNAc-RNA delivery service, representing the apex of liver-targeted oligonucleotide technology. This service is designed for therapeutic programs demanding maximum potency and the lowest possible effective dose. Building upon the established clinical success of multivalent GalNAc systems, our proprietary tetra-antennary ligand features four GalNAc moieties precisely arranged to engage the multimeric aasialoglycoprotein receptor (ASGPR) on the surface of liver hepatocytes with ultra-high avidity. The ASGPR is a C-type lectin that acts as a dedicated liver gatekeeper, and early biological studies indicate that four binding residues can maximize affinity compared to lower valency options. Our tetra-antennary platform ensures superior and more sustained gene silencing via RNA interference (RNAi) or antisense oligonucleotide (ASO) mechanisms. By delivering an ultra-concentrated payload exclusively to the hepatocyte cytoplasm via highly efficient, receptor-mediated endocytosis, CD BioGlyco provides the chemical and biological foundation for long-acting, low-dose therapeutics.
Core Technologies
The power of the tetra-antennary system lies in the strategic design of its three molecular components: the targeting ligand, the conjugation scaffold, and the therapeutic payload.
- Ultra-High Avidity Tetra-antennary Ligand
The ASGPR exists on the hepatocyte surface as a multimeric complex, comprising both major (ASGR1) and minor (ASGR2) subunits. This multimeric structure contains multiple binding sites, which our tetra-antennary ligand is specifically engineered to bridge. By presenting four GalNAc units in an optimal spatial arrangement, we ensure a super-selective cooperative binding effect—or avidity—that is critical for maximal internalization efficiency. The binding of the GalNAc ligand to ASGPR is highly dependent on both the number and the geometry of the sugar residues, and our tetra-antennary cluster is optimized to achieve the absolute highest affinity for the receptor complex, translating directly to maximum cellular uptake and superior in vivo potency.
- Custom Scaffold and Linker Engineering
The synthesis of a high-valency ligand is complex; success hinges on the central scaffold used to present the four GalNAc units. We utilize proprietary, chemically robust scaffolds designed for structural rigidity and optimal presentation geometry. Furthermore, the linker connecting this tetra-antennary cluster to the oligonucleotide payload is engineered to facilitate efficient endosomal escape. The delicate balance of stability in circulation and the ability to dissociate from the ASGPR within the endosome for cytoplasmic release is maximized through our refined chemical linker technology.
- Advanced Oligonucleotide Chemical Stabilization
A highly potent targeting system requires an equally robust payload. We incorporate state-of-the-art chemical modifications into the oligonucleotide (siRNA or ASO) to maximize stability, minimize off-target effects, and enhance affinity for the target mRNA. Essential modifications include:
- Phosphorothioate (PS) linkages: Introduced into the backbone to provide protection against circulating nucleases, dramatically improving the in vivo half-life.
- 2'-Sugar modifications (2'-F, 2'-OMe): Strategically placed on the ribose sugar to enhance metabolic stability, improve duplex thermal stability, and tune the activity profile of the RNA payload.
Unlocking Ultra-Potency: Tetra-antennary GalNAc-RNA Delivery
We offer comprehensive solutions tailored to the stringent demands of high-potency drug development:
- Custom Conjugate Scaffold Selection
We offer a library of proprietary tetra-antennary scaffold designs, allowing you to select or collaboratively develop the optimal cluster geometry and linker length for your specific therapeutic mechanism (siRNA, ASO, or microRNA inhibitors).
- Scale-Up
Production is available from milligram discovery scale up to multi-kilogram commercial scale. Our standardized, modular synthesis approach ensures seamless and predictable scale-up.
- Oligonucleotide Chemistry Flexibility
Support for all major chemical stabilization strategies, including S-c-ET, LNA, C-type, and BNA modifications, in addition to the standard PS and 2'-modifications, ensuring compatibility with cutting-edge oligonucleotide payloads.
Workflow
Ligand Design & Validation
In silico modeling and chemical design of the tetra-antennary scaffold geometry and linker chemistry to confirm optimal spatial orientation for maximum ASGPR binding avidity. The target sequence (siRNA/ASO) is simultaneously optimized for biological efficacy and chemical stability.
Tetra-antennary Ligand Synthesis
Multi-step organic synthesis of the proprietary tetra-antennary GalNAc cluster, culminating in the preparation of a highly purified, activated phosphoramidite or conjugation-ready PFP-ester intermediate.
High-Fidelity Oligonucleotide Synthesis
Automated solid-phase synthesis of the chemically modified oligonucleotide (ASO or siRNA strands) at the required scale, ensuring all necessary backbone (PS) and sugar (2'-F/OMe) modifications are precisely incorporated.
Covalent Conjugation & Deprotection
High-efficiency coupling of the activated tetra-antennary ligand to the oligonucleotide, typically at the 3' or 5' terminus. This is followed by cleavage from the solid support and primary deprotection of the nucleobases and phosphates.
Multi-Stage Purification
Rigorous, multi-pass purification using advanced chromatography techniques (e.g., anion exchange and reverse phase HPLC) to isolate the final tetra-antennary conjugate. This demanding step is critical for removing truncated sequences and unreacted ligand to achieve the required high purity (>95%).
Comprehensive Quality Assurance (QA/QC)
Detailed analytical characterization, including high-resolution mass spectrometry (HRMS) for confirmation of exact mass, analytical HPLC for purity and homogeneity assessment, and final formulation checks (e.g., Endotoxin testing) for preclinical use.
Publication Data
Journal: Molecules
DOI: 10.3390/molecules28207184
Published: 2023
IF: 4.6
Results: In this study, the authors introduced an innovative dithiomaleimide (DTM)-based tetra-antennary GalNAc conjugate designed to enhance siRNA delivery while providing inherent traceability. The conjugate was efficiently synthesized through a series of click-type reactions—amidations, thiol-dibromomaleimide addition, and copper-catalyzed azide-alkyne cycloaddition (CuAAC)—resulting in a structure that demonstrated siRNA delivery efficiency comparable to the standard GalNAc L96 when targeting mouse transthyretin (mTTR). Crucially, the internal DTM moieties emitted strong fluorescence, allowing real-time tracking of cellular uptake and distribution without incorporating external fluorophores, thereby reducing costs and minimizing potential side effects. This "two-birds-with-one-stone" approach combines robust gene silencing capabilities with self-monitoring features, offering significant promise for advancing RNAi therapeutics by simplifying formulation and improving safety profiles in gene therapy applications.
Applications
Chronic Viral Hepatitis
Developing next-generation antivirals aimed at silencing key viral replication components within the hepatocyte, where the high potency of the tetra-antennary system can drive deep, sustained reductions in viral load necessary for a functional cure.
Severe Genetic Metabolic Disorders
Treating rare, life-threatening conditions like primary hyperoxaluria (PH) or acute hepatic porphyria (AHP), where even small variations in gene expression are catastrophic. Our system ensures the robust and reliable target suppression needed for these critical indications.
Atherosclerotic Cardiovascular Risk Reduction
Targeting genes responsible for key circulating proteins synthesized in the liver, such as ANGPTL3 or Lp(a). The ability to achieve ultra-low ED50 is critical for developing high-volume, cost-effective therapies for large patient populations.
Transthyretin (TTR) Amyloidosis
Silencing the TTR gene to stop the production of the misfolded protein that causes systemic amyloidosis. Our platform is optimized to maintain the potent, sustained knockdown necessary to halt disease progression and manage chronic symptoms.
Advantages
Maximum Target Avidity and Potency
By leveraging four GalNAc units, we achieve maximal binding affinity for the ASGPR receptor multimer, often resulting in sub-nanomolar binding, which is essential for maximizing cellular internalization and achieving the lowest possible effective dose in in vivo models.
Extended Duration of Effect
The enhanced uptake efficiency means a larger fraction of the therapeutic dose is captured by hepatocytes. This higher initial concentration leads to a more robust and prolonged gene silencing effect, supporting significantly extended dosing intervals compared to lower-valency systems.
Highest Assurance of Liver Specificity
The cooperative binding provided by the quadrivalent cluster drives super-selective recognition of the ASGPR, which is uniquely abundant on hepatocytes (up to 500,000 receptors per cell), ensuring delivery fidelity and virtually eliminating off-target effects in non-hepatic tissues.
Robust SAR-Driven Design
Our tetra-antennary scaffolds are designed based on deep structure-activity relationship (SAR) data, focusing not just on the number of ligands, but the precise geometric spacing required to simultaneously engage multiple ASGPR binding domains for maximal efficacy.
Frequently Asked Questions
Customer Review
We chose CD BioGlyco's tetra-antennary platform for our high-impact target, hoping to push the boundaries of in vivo potency. The results were astounding—we achieved therapeutic knockdown at a dose that was significantly lower than the established tri-GalNAc literature benchmark. The synthesis was complex, but the quality and documentation were flawless.
— Dr. K. Patel, Senior Principal Scientist, Cardiovascular Research
The transition to the tetra-antennary chemistry with CD BioGlyco was smooth, thanks to their dedicated project team. Their ability to consistently manufacture a chemically-defined, highly pure quadrivalent ligand at the gram scale is genuinely impressive and has de-risked our commercialization pathway substantially.
— Manager, E, Head of Bioconjugation, Oligonucleotide Development
We value simplicity in manufacturing. While the chemistry is advanced, their final product documentation and high-purity yield simplified our downstream formulation and QC immensely. The material was ready for injection with minimal additional processing—a significant time saver.
— Process Engineer, M., R&D Operations Group,
Associated Services
At CD BioGlyco, the tetra-antennary GalNAc-RNA delivery service is not merely a component—it is a complete, ultra-potent solution for the development of best-in-class liver-targeted nucleic acid therapeutics. By combining sophisticated quadrivalent chemistry with rigorous quality control and flexible production scale, we empower you to achieve unparalleled potency and the favorable dosing profiles required for commercial success. Leverage our expertise to minimize dose, maximize duration, and accelerate your path to the clinic. If you want to translate your scientific breakthroughs into high-impact medicines, contact us!
Reference
- Kong, S.; 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. (Open Access)