Abstract
This study highlights that small geometric constraints with the GalNAc moiety can tune receptor engagement and serum stability. The ribofuranose ring is a 2′-O-methyl modification, used in the siRNA backbone to improve metabolic stability and reduce nuclease susceptibility.8 The constrained ribofuranose conformation introduced into GalNAc increased serum stability and hepatic parenchymal clearance, thereby increasing systemic exposure. They also demonstrate the kilogram-scale synthesis of the G5 GalNAc-CPG support, making it feasible for manufacturing. PCSK9 is an ideal benchmark target as it is clinically validated, and the observed performance can be applied to other hepatocyte-expressed genes. The inc-G5 GalNAc has progressed to phase 1 trials in China for PCSK9- and AGT-targeting siRNA. In a non-human primate study, inc-G5 GalNAc-siRNA conjugates showed a safety profile consistent with established GalNAc-siRNA (inc-L96) conjugates. A comparison of toxicology in AGT-targeting siRNA versus zilebesiran showed similar clinical chemistry and histopathology, supporting inc-G5 GalNAc as a potent, clinically translatable GalNAc delivery scaffold.
Main text
N-acetylgalactosamine (GalNAc) conjugation has emerged as a leading strategy for hepatocyte-targeted delivery, accelerating clinical translation of oligonucleotide therapeutics. GalNAc binds to the asialoglycoprotein receptor (ASGPR) on hepatocytes with high affinity, triggering receptor-mediated endocytosis.1 It has been successfully demonstrated that GalNAc-small interfering RNA (siRNA) delivery enables effective in vivo hepatocyte-targeted gene silencing.2 This platform yielded the first FDA-approved GalNAc-siRNA therapeutic (givosiran) in 2019 for the treatment of acute hepatic porphyria (AHP) and has since expanded to nine additional FDA-approved GalNAc-conjugated oligonucleotide therapies. Givosiran consists of a branched triantennary GalNAc L96 ligand, conjugated at the 3′ terminus of the siRNA strand with a trans-4-hydroxyprolinol linker and a C12 spacer. Incremental changes in the chemistry of the GalNAc L96 ligand can yield improved hepatic selectivity, enhanced pharmacokinetics, and easier manufacturing.1 The specific spatial arrangement and geometry of the GalNAc sugar enhances avidity with trivalent GalNAc, demonstrating ∼1,000-fold higher affinity than divalent or monovalent ligands.3 The in vivo performance of GalNAc is governed by the properties of the chemical linker and the linkage chemistry between the siRNA and GalNAc. GalNAc-siRNA conjugates are synthesized during solid-phase synthesis either by using a GalNAc-preloaded solid support (3′ conjugation) or by incorporating a GalNAc-phosphoramidite unit (5′ conjugation). To improve the scalability, solution-phase conjugation strategies that include click chemistry, amide coupling, and maleimide thiol reactions have been employed.4 The commonly used linker for solid-phase synthesis of GalNAc-siRNA conjugates is trans-4-hydroxyprolinol. The trifunctional trans-4-hydroxyprolinol scaffold enables simultaneous ligand conjugation via its secondary amine to a solid-phase support and via its primary/secondary hydroxyl groups2 to an oligonucleotide. The selection of the linker is governed by its structure-function relationships and its compatibility with solid-phase synthesis. A study exploring linker activity has shown improved efficacy when nitrogen used for ligand conjugation was incorporated into the cyclic ring structure, compared with the GalNAc L96 ligand. The potency of siRNA knockdown was significantly reduced in acyclic or non-ring motifs.5 Studies have also shown that bio-cleavable linkers did not improve intracellular potency and can reduce plasma stability.6 These structure-function dependencies motivate comparative analysis in which the GalNAc linker chemistry is modified to quantify effects on silencing, biodistribution, and pharmacokinetics.
In this issue of Molecular Therapy Nucleic Acids, Huang et al. synthesized novel GalNAc solid supports incorporating deoxyribofuranose (inc-G1) and ribofuranose (inc-G5) ring moieties into the GalNAc scaffold and compared their performance with the approved GalNAc-siRNA construct containing a trans-4-hydroxyprolinol ring moiety (inc-L96).7 This strategy builds upon prior work demonstrating that ribofuranose incorporation in the nucleic acid backbone increases metabolic stability and hepatic accumulation.8 In this study, Huang et al. compared the activity of various GalNAc derivatives by conjugating a panel of siRNAs targeting PCSK9 mRNA and summarized their relative silencing efficacy, biodistribution, and pharmacokinetic properties. In humanized PCSK9 mice, a single subcutaneous dose (6 mg/kg) resulted in a significant difference in PCSK9 knockdown across different GalNAc-siRNA conjugates. At day 14, inc-G5 and inc-G1 reduced PCSK9 serum levels to 4.4% and 20.2%, respectively, with inc-G5 showing a 10.2% greater PCSK9 reduction than the trans-4-hydroxyprolinol ring (inc-L96). Further, in the liver, inc-G5 exhibited an 11.1% greater reduction in PCSK9 mRNA than the trans-4-hydroxyprolinol ring (inc-L96). The authors explored the in vivo biodistribution of all three GalNAc-siRNA conjugates labeled with Cy5 via subcutaneous injection. Biodistribution profiles showed that GalNAc with the ribofuranose ring (inc-G5) showed a higher accumulation in the liver, whereas deoxyribofuranose (inc-G1) and trans-4-hydroxyprolinol ring (inc-L96) conjugates showed preferential renal retention. Pharmacokinetic analysis from sampling rat tissue showed that GalNAc with ribofuranose (inc-G5) achieved a longer elimination half-life (t1/2), higher peak plasma concentration (Cmax), and the largest area under the curve (AUC0–last) than GalNAc with deoxyribofuranose (inc-G1) and trans-4-hydroxyprolinol ring (inc-L96) conjugates (Table 1). Taken together, these results demonstrate that inc-G5 increases liver residence time, reduces renal elimination, and achieves more effective PCSK9 knockdown (Figure 1).
Table 1.
Comparative summary of PCSK9 suppression, tissue distribution, and pharmacokinetic profiles for inc-G1, inc-G5, and inc-L96
| Study parameter | Metric/tissue | inc-G1 | inc-G5 | inc-L96 | |
|---|---|---|---|---|---|
| Humanized PCSK9 mice (6 mg/kg, sc) | Serum PCSK9 protein remaining | ∼20.2% | ∼4.4% | ∼10.2% | |
| Hepatic mRNA level remaining | ∼25% | ∼8.8% | ∼19.9% | ||
| Biodistribution: BALB/c mice (fluorescence imaging, 6 mg/kg, sc) | 4 h | liver | 6.00E+09 | 9.12E+09 | 8.22E+09 |
| kidney | 7.32E+09 | 4.31E+09 | 9.01E+09 | ||
| liver-to-kidney ratio | 0.82 | 2.12 | 0.91 | ||
| 8 h | liver | 7.42E+09 | 9.16E+09 | 5.99E+09 | |
| kidney | 9.19E+09 | 3.44E+09 | 9.15E+09 | ||
| liver-to-kidney ratio | 0.81 | 2.66 | 0.65 | ||
| PK: Sprague-Dawley rats (5 mg/kg, sc) | T1/2 (h) | liver | 56 | 110 | 74 |
| kidney | 155 | 164 | 115 | ||
| Cmax (ng/mL) | liver | 32,100 | 44,500 | 35,367 | |
| kidney | 1,423 | 1,643 | 3,590 | ||
| AUC0–last (h.ng/mL) | liver | 2,184,569 | 3,394,662 | 2,505,198 | |
| kidney | 311,997 | 257,567 | 435,510 | ||
| liver-to-kidney ratio | 7.00 | 13.18 | 5.75 | ||
Across three animal models (humanized mice, BALB/c mice, and SD rats), inc-G5 exhibits the greatest reduction in PCSK9 protein and mRNA levels, consistent with its higher liver-to-kidney distribution ratio and increased hepatic exposure (Cmax and AUC0–last) relative to inc-G1 and inc-L96.
Figure 1.
Structural optimization of GalNAc-ASO conjugates enhances hepatic delivery and PCSK9 silencing
Schematic comparison of an approved GalNAc-antisense oligonucleotide (ASO) conjugate (inc-L96) with next-generation modified ribosugar GalNAc-ASO conjugates (inc-G1 and inc-G5). Structural modifications include replacement of the tetrahydropyrrole linker backbone with deoxyribofuranose or ribofuranose ring skeletons, designed to improve receptor engagement and in vivo stability. GalNAc conjugation enables selective uptake via the asialoglycoprotein receptor (ASGPR) on hepatocytes, leading to efficient intracellular delivery of the antisense strand. Binding of the ASO to PCSK9 mRNA promotes transcript degradation and reduces PCSK9 protein production. Comparative in vivo data illustrate enhanced liver specificity and the high level of PCSK9 knockdown by inc-G5 among the tested conjugates.
This study highlights that small geometric constraints with the GalNAc moiety can tune receptor engagement and serum stability. The ribofuranose ring is a 2′-O-methyl modification, used in the siRNA backbone to improve metabolic stability and reduce nuclease susceptibility.8 The constrained ribofuranose conformation introduced into GalNAc increased serum stability and hepatic parenchymal clearance, thereby increasing systemic exposure. They also demonstrate the kilogram-scale synthesis of the G5 GalNAc-CPG support, making it feasible for manufacturing. PCSK9 is an ideal benchmark target as it is clinically validated, and the observed performance can be applied to other hepatocyte-expressed genes. The inc-G5 GalNAc has progressed to phase 1 trials in China for PCSK9- and AGT-targeting siRNA. In a non-human primate study, inc-G5 GalNAc-siRNA conjugates showed a safety profile consistent with established GalNAc-siRNA (inc-L96) conjugates. A comparison of toxicology in AGT-targeting siRNA versus zilebesiran showed similar clinical chemistry and histopathology, supporting inc-G5 GalNAc as a potent, clinically translatable GalNAc delivery scaffold.
Acknowledgments
This work was supported by the UConn Startup grant to R.B. All figures were created using BioRender.com.
Author contributions
R.B., S.P.P., and R.R. wrote and edited the commentary.
Declaration of interests
The authors declare no competing interests.
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