siRNA(Oligo Drugs) Study: Role of GalNAc-siRNA Conjugates in Lysosomal Stability and siRNA Delivery for Overcoming siRNA Escape

siRNA(Oligo Drugs) Study: Role of GalNAc-siRNA Conjugates in Lysosomal Stability and siRNA Delivery for Overcoming siRNA Escape

Keywords: GalNAc-siRNA, siRNA Delivery, siRNA Escape, Liver Lysosomes, Hepatocyte Lysosomes, Tritosome, Lysosome Catabolism, Lysosomal Stability, Lysosomal Acid Phosphatase

IPHASE Product

Introduction
RNA-based therapeutics have emerged as a transformative approach in treating various diseases through targeted gene silencing. Among these treatments, siRNA drugs are gaining attention for their improved specificity and efficacy. A major challenge in siRNA Delivery is ensuring efficient siRNA escape from the endocytic pathway before degradation in liver lysosomes and Hepatocyte lysosomes. In vitro studies increasingly focus on how siRNA formulations promote siRNA escape while interacting with cellular components. This interaction involves critical factors such as lysosome catabolism, lysosomal stability, and degradation by Lysosomal acid phosphatase. Optimizing these parameters is essential for enhancing siRNA Delivery and achieving effective siRNA escape.

siRNA Delivery and Lysosomal Entrapment
Effective siRNA Delivery to hepatocytes often relies on carriers such as lipid nanoparticles (LNPs) or conjugates like GalNAc-siRNA, which target liver-specific receptors. Despite these innovations, a significant fraction of siRNA is trafficked to liver lysosomes and Hepatocyte lysosomes, where rapid lysosome catabolism leads to degradation. The acidic environment, enriched with Lysosomal acid phosphatase, challenges lysosomal stability and hinders siRNA escape. To improve therapeutic outcomes, research on siRNA is focusing on enhancing siRNA escape from these lysosomal compartments, thereby improving overall siRNA Delivery.

Mechanism of GalNAc-siRNA Conjugates 

GalNAc-siRNA conjugates are a promising approach for siRNA delivery, leveraging the high specificity of N-acetylgalactosamine (GalNAc) binding to asialoglycoprotein receptors (ASGPR) on hepatocytes. This interaction facilitates rapid endocytosis, allowing siRNA to enter liver cells efficiently. Upon uptake, the conjugates are internalized via clathrin-coated pits, released into the cellular lumen, and subsequently activate RNA interference (RNAi) by dissociating from their sialyl-GalNAc linkers.

To enhance the stability and therapeutic efficacy of GalNAc-siRNA conjugates, several chemical modifications are employed:

• 2'-F and 2'-OMe Modifications – These modifications prevent RNase degradation while maintaining compatibility with RNA interference (RNAi) machinery, mimicking the biophysical properties of the natural 2'-OH group.

• Phosphorothioate Modifications – Adding phosphorothioate groups at the 5' and 3' ends of siRNA strands increases potency, stability, and RNAi durability in vivo.

• Optimized RNAi Triggers – Common siRNA designs include the 21/21 nucleotide template with 19 base pairs and 3' overhangs, and the 21/23 nucleotide template, which has a blunt 5' guide strand end and a 3' overhang. These optimizations improve siRNA effectiveness and longevity.

Lysosomal Barriers
The journey of siRNA from administration to its site of action in hepatocytes is fraught with obstacles, especially the sequestration and degradation within liver lysosomes and Hepatocyte lysosomes. Aggressive lysosome catabolism in these compartments, driven in part by enzymes like Lysosomal acid phosphatase, compromises lysosomal stability and limits siRNA escape. Overcoming these lysosomal barriers is critical for successful siRNA Delivery. Advances in -siRNA technology focus on modulating lysosome catabolism to enhance lysosomal stability and promote more efficient siRNA escape from both liver lysosomes and Hepatocyte lysosomes.

Lysosome Catabolism and siRNA Degradation
In hepatocytes, lysosome catabolism is a major barrier to the stability of therapeutic siRNA. Within the acidic milieu of liver lysosomes and Hepatocyte lysosomes, enzymatic activities—including those of Lysosomal acid phosphatase—accelerate siRNA degradation. This degradation compromises lysosomal stability and reduces the window for effective siRNA escape. Recent studies on siRNA formulations have demonstrated that modulating Lysosomal acid phosphatase activity can mitigate lysosome catabolism, thereby preserving siRNA integrity and enhancing siRNA Delivery and siRNA escape.

Utilizing Tritosome Models in siRNA Research
In addition to conventional lysosomal studies, isolated tritosomes offer an advanced model for evaluating lysosomal behavior. Specifically, rat liver tritosomes—which are hepatic lysosomes loaded with non-ionic surfactants—have been employed as a predictive in vitro system to study lysosome catabolism and membrane stability. These tritosome models enable researchers to closely mimic and quantify the enzymatic degradation processes that impact siRNA stability. By incorporating insights from rat liver tritosome studies, scientists can better optimize formulation strategies to enhance siRNA escape, ultimately contributing to more effective RNA-based therapeutics.

Metabolic research system and selection of oligonucleotides

As with traditional small molecule drugs, siRNA formulations require comprehensive in vitro metabolic stability studies during preclinical development. These studies evaluate the impact of lysosome catabolism and the role of Lysosomal acid phosphatase in degrading siRNA within liver lysosomes and Hepatocyte lysosomes. Emphasis is placed on optimizing siRNA Delivery and ensuring robust siRNA escape. Various test systems—such as liver homogenates, isolated liver lysosomes, and primary hepatocytes—are employed to mimic the hepatic environment. Enhancing lysosomal stability through these assessments is key to improving the performance of siRNA drugs.

Conclusion:

siRNA therapeutics are transforming precision medicine by enabling targeted gene silencing, though challenges like lysosomal degradation remain. Acidic lysosomes and enzymes such as lysosomal acid phosphatase hinder siRNA stability, but innovations like GalNAc-siRNA conjugates and chemical modifications (e.g. 2’-F/2’-OMe, phosphorothioate) improve durability and lysosomal escape. In vitro studies with liver models further optimize these formulations, paving the way for more effective RNA-based treatments.

With years of R&D experience, IPHASE has launched high-end scientific research reagents in many fields and types, providing screening tools for early drug development, new materials, new methods and new means for the exploration of the field of life sciences, and convenient products for genotoxicity research of food, pharmaceuticals, chemicals, etc.