Click-based GalNac-RNA Delivery Service
The clinical success of RNA therapeutics is fundamentally tethered to the efficiency of tissue-specific delivery. While lipid nanoparticles (LNPs) have revolutionized the field, the emergence of ligand-conjugated oligonucleotides offers a "lock-and-key" precision that maximizes potency while minimizing systemic toxicity. CD BioGlyco specializes in click chemistry-based GalNAc-RNA delivery services, leveraging the highly efficient asialoglycoprotein receptor (ASGPR) pathway. By utilizing bioorthogonal click chemistry, we achieve site-specific, high-yield conjugation of trivalent N-acetylgalactosamine (GalNAc) clusters to your therapeutic RNA. This platform is specifically designed to overcome the pharmacokinetic hurdles of "naked" oligonucleotides, ensuring robust internalization into hepatocytes for durable gene silencing or modulation.
Our platform integrates the Nobel Prize-winning principles of click chemistry with advanced carbohydrate engineering to deliver a superior targeting vehicle:
We utilize copper-free click chemistry with dibenzocyclooctyne (DBCO) moieties. This biorthogonal reaction occurs under mild, physiological conditions without the need for toxic metal catalysts, preserving the integrity of sensitive RNA species.
Our proprietary trivalent ligands are engineered to mirror the natural stoichiometry of the trimeric ASGPR. This multivalent interaction significantly enhances binding avidity (nanomolar affinity) compared to monovalent designs, facilitating rapid endocytosis.
We combine click conjugation with strategic 2'-O-methyl (2'-OMe) and 2'-deoxy-2'-fluoro (2'-F) modifications. This dual approach protects the conjugate from exonuclease degradation and ensures a long-lasting intracellular reservoir.
Our service offers a range of customizable linkers, including PEG-based spacers for solubility and bioreversible (cleavable) bonds that optimize the release of the RNA payload within the endosomal compartment.
The click chemistry-based GalNAc-RNA delivery service provides a comprehensive solution for researchers transitioning from discovery to pre-clinical development. Our scope encompasses the custom synthesis of a wide array of oligonucleotide modalities, including siRNA, antisense oligonucleotides (ASOs), microRNAs (miRNAs), and even larger aptamers. We offer unprecedented flexibility in conjugation sites, allowing for 5'-end, 3'-end, or multiple internal GalNAc attachments to optimize the therapeutic index for each specific sequence.
Our service is built on a modular platform that supports various scale requirements, from milligram quantities for initial screening to multigram scales for toxicology and NHP studies. Beyond simple conjugation, our scope includes the implementation of enhanced stability chemistry (ESC) and Advanced ESC designs, which are critical for achieving the "months-long" duration of effect observed in modern clinical candidates. We also provide specialized linker design services, offering PEGylated or alkyl spacers to fine-tune the hydrophobicity and bioavailability of the final conjugate. With us, you gain access to a fully integrated pipeline that handles everything from nucleotide synthesis and click chemistry to advanced LC-MS characterization and impurity profiling.
We conduct a comprehensive analysis of your target RNA (siRNA, ASO, or miRNA) to determine the optimal sites for functionalization, ensuring minimal impact on its biological activity and stability. The synthesis is then performed using solid-phase techniques, precisely incorporating azide groups at predetermined positions, such as the 5' or 3' terminus, or at internal sites—via modified phosphoramidite chemistry. This site-specific modification is critical for enabling subsequent bioorthogonal conjugation while preserving the structural integrity of the RNA molecule.
The azide-functionalized RNA undergoes a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction with our custom-synthesized, trivalent GalNAc-DBCO cluster. This copper-free click chemistry approach is conducted under controlled, mild conditions to maximize yield and maintain RNA integrity. We employ real-time monitoring with analytical techniques such as UV spectroscopy or HPLC to track the reaction progress, ensuring quantitative conversion of the RNA and achieving precise stoichiometric control over the final conjugate.
Following conjugation, the crude product is subjected to a stringent, multi-step purification process. We primarily utilize preparative-scale reverse-phase HPLC (RP-HPLC) or ion-exchange chromatography (IEX), selected based on the physicochemical properties of the specific conjugate. This step effectively separates and removes unreacted ligands, excess reagents, and any trace impurities, consistently yielding conjugates with pharmaceutical-grade purity levels exceeding 98%.
Every batch of the final conjugate undergoes a rigorous battery of quality control tests. This includes liquid chromatography-mass spectrometry (LC-MS) for exact mass verification and confirmation of identity, as well as analytical size-exclusion chromatography (SEC) to assess monodispersity, detect any potential aggregates, and ensure the product meets predefined specifications for homogeneity and stability.
As an optional service, we can perform targeted in vitro assays to functionally validate the conjugate. These studies typically include surface plasmon resonance (SPR)-based ASGPR-binding assays to quantify targeting ligand affinity, and cell-based uptake studies using relevant hepatocyte models to demonstrate targeted delivery efficiency. This provides critical data on the biological performance of the final product before shipment.
Journal: Molecules
DOI: 10.3390/molecules28207184
IF: 4.6
Published: 2023
Results: This research article presents the development of a novel dithiomaleimide (DTM)-based tetra-antennary GalNAc conjugate designed to enable both efficient siRNA delivery and intrinsic traceability without incorporating external fluorophores. The authors successfully constructed this "two-birds-with-one-stone" system using a series of efficient click-type reactions, including amidations, thiol-dibromomaleimide addition, and CuAAC. The resulting conjugate demonstrated comparable siRNA delivery efficiency to the standard GalNAc-L96 when targeting mouse transthyretin (mTTR), as validated through cellular uptake studies and gene silencing assays in primary mouse hepatocytes. Crucially, the incorporated DTM moieties exhibited strong intrinsic fluorescence, providing a built-in tracking capability that eliminates the need for additional dye molecules, thereby reducing potential side effects and synthesis costs. The study confirms that this innovative approach maintains effective delivery performance while offering enhanced traceability, presenting a promising strategy for advancing RNAi therapeutics by combining delivery functionality and monitoring capabilities within a single molecular entity.
Chronic Hepatitis B (HBV)
We enable the targeted delivery of antiviral RNA molecules to hepatocytes, where they can silence viral transcripts and potentially lead to functional cure by reducing HBsAg levels effectively.
Transthyretin (ATTR) Amyloidosis
Our click-conjugated siRNAs can precisely target the liver to inhibit the production of TTR protein, addressing the root cause of both hereditary and wild-type amyloid polyneuropathy and cardiomyopathy.
Primary Hyperoxaluria
By delivering siRNAs that silence hydroxyacid oxidase 1 (HAO1), our platform helps reduce hepatic oxalate production, preventing kidney stones and systemic oxalosis in patients with genetic enzyme deficiencies.
Acute Hepatic Porphyrias
We provide the delivery tools to target ALAS1 mRNA in hepatocytes, effectively managing the overproduction of neurotoxic intermediates and preventing the debilitating attacks associated with this rare metabolic disease.
Bioorthogonal Precision
Our click chemistry approach ensures that the conjugation reaction only occurs at the designated azide/alkyne sites. This site-specificity eliminates the "messy" mixture of products often found with traditional NHS-ester or maleimide chemistries.
Copper-Free Safety
By utilizing SPAAC (strain-promoted) technology, we avoid the use of copper catalysts. This is critical for biological applications, as it prevents oxidative damage to the RNA and eliminates potential metal toxicity in downstream in vivo studies.
Rapid Internalization Kinetics
The high-affinity trivalent GalNAc ligands we use are rapidly recognized by ASGPR. This leads to the internalization of over a million RNA molecules every 15 minutes per cell, far exceeding the threshold required for therapeutic effect.
Superior Stability in Acidic Media
Our conjugates are engineered to remain stable during endosomal trafficking. Maintaining conjugate integrity within acidic intracellular compartments is the key driver of long-term pharmacological activity.
CD BioGlyco's click chemistry platform is a game-changer. The purity of the GalNAc-siRNA conjugates we received was exceptional, and the in vivo potency in our NASH model exceeded all previous benchmarks.
— By Dr. J.H., Director of RNA Discovery
We struggled with copper toxicity using other providers. Switching to CD BioGlyco's SPAAC-based service eliminated our cell viability issues and allowed us to focus on the biology of our target.
— By Manager, Metabolic Research Unit
The team at CD BioGlyco provided incredible support in designing the right linker for our ASO project. Their 5'-trivalent GalNAc conjugates showed a 5-fold improvement in liver uptake.
— By Dr. S.L., Senior Principal Scientist
High-purity, custom siRNA synthesis with a wide range of chemical modifications for maximum stability.
Specialized synthesis of microRNA mimics and inhibitors tailored for gene regulation studies.
Advanced antisense oligonucleotide production featuring phosphorothioate and 2'-MOE modifications.
CD BioGlyco is your premier partner for precision RNA delivery. Our click chemistry-based GalNAc-RNA platform represents the pinnacle of site-specific hepatocyte targeting, offering a robust, scalable, and highly potent solution for your therapeutic needs. From initial design to large-scale synthesis, we provide the expertise and quality control necessary to drive your liver-directed programs forward. Contact us!
Reference
