Evaluating the oral delivery of GalNAc-conjugated siRNAs in rodents and non-human primates.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

Recovery of chronic motor neuropathy due to acute intermittent porphyria after givosiran treatment in a young boy: a case report.

BACKGROUND: We describe the first case of a pediatric patient with acute intermittent porphyria and severe chronic porphyric neuropathy treated with givosiran, a small-interfering RNA that drastically decreases delta-aminolevulinic acid production and reduces porphyric attacks' recurrence. CASE REPORT: A 12-year-old male patient with refractory acute intermittent porphyria and severe porphyric neuropathy was followed prospectively for 12 months after givosiran initiation (subcutaneous, 2.5 mg/kg monthly). Serial neurological, structural, and resting-state functional magnetic resonance imaging (MRI) evaluations were performed, including clinical scales and neurophysiological tests. Delta-aminolevulinic acid urinary levels dropped drastically during treatment. In parallel, all the administered neurological rating scales and neurophysiological assessments showed improvement in all domains. Moreover, an improvement in central motor conduction parameters and resting-state functional connectivity in the sensory-motor network was noticed. At the end of the follow-up, the patient could walk unaided after using a wheelchair for 5 years. CONCLUSIONS: A clear beneficial effect of givosiran was demonstrated in our patient with both clinical and peripheral nerve neurophysiologic outcome measures. Moreover, we first reported a potential role of givosiran in recovering central motor network impairment in acute intermittent porphyria (AIP), which was previously unknown. This study provides Class IV evidence that givosiran improves chronic porphyric neuropathy.

Characterization of Ssc, an N-acetylgalactosamine-containing Staphylococcus aureus surface polysaccharide.

Whole genome sequencing has revealed that the genome of Staphylococcus aureus possesses an uncharacterized 5-gene operon (SAOUHSC_00088-00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus as ssc for staphylococcal surface carbohydrate. We found that the ssc genes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed in Escherichia coli and extracted by heat treatment. Ssc was also conjugated to AcrA from Campylobacter jejuni in E. coli using protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting of N-acetylgalactosamine. We further demonstrated that the expression of the ssc genes in S. aureus affected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCE: Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide.

Genetic and functional diversity of ß-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis.

ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

Efficient Tandem Copper-Catalyzed Click Synthesis of Multisugar-Modified Oligonucleotides.

Nucleic acids in the form of siRNA, antisense oligonucleotides or mRNA are currently explored as new promising modalities in the pharmaceutical industry. Particularly, the success of mRNA-vaccines against SARS-CoV-2, along with the successful development of the first sugar-modified siRNA therapeutics has inspired the field. The development of nucleic acid therapeutics requires efficient chemistry to link oligonucleotides to chemical structures that can improve stability, boost cellular uptake, or enable specific targeting. For the siRNA therapeutics currently in use, modification of the 3'-end of the oligonucleotides with triple-N-acetylgalactosamine (GalNAc)3 was shown to be of significance. This modification is currently achieved through cumbersome multistep synthesis and subsequent loading onto the solid support material. Herein, we report the development of a bifunctional click-reactive linker that allows the modification of oligonucleotides in a tandem click reaction with multiple sugars, regardless of the position within the oligonucleotide, with remarkable efficiency and in a one-pot reaction.

Identification and characterization of a PL35 GAGs lyase with 4-O-sulfated N-acetylgalactosamine (A-type)-rich structures producing property.

Glycosaminoglycan (GAG) lyases are important tools for investigating the structure of GAGs and preparing low-molecular-weight GAGs. The PL35 family, a recently established polysaccharide lyase family, should be further investigated. In this study, we discovered a new GAG lyase, CHa1, which belongs to the PL35 family. When expressed heterologously in Escherichia coli (BL21), CHa1 exhibited high expression levels and solubility. The optimal activity was observed in Tris-HCl buffer (pH 7.0) or sodium phosphate buffer (pH 8.0) at 30 °C. The specific activities towards HA, CSA, CSC, CSD, CSE, and HS were 3.81, 13.03, 36.47, 18.46, 6.46, and 0.50 U/mg protein, respectively. CHa1 digests substrate chains randomly that acting as an endolytic lyase and shows a significant preference for GlcA-containing structures, prefers larger oligosaccharides (≥UDP8) and can generate a series of oligosaccharides composed mainly of the A unit when digesting CSA. These oligosaccharides include ΔC-A, ΔC-A-A, ΔC-A-A-A, ΔC-A-A-A-A, and ΔC-A-A-A-A-A. The residues Tyr257 and His421 play crucial roles in the catalytic process, and Ser211, Asn212, Asn213, Trp214, Gln216, Lys360, Arg460 and Gln462 may participate in the binding process of CHa1. This study on CHa1 contributes to our understanding of the PL35 family and provides valuable tools for investigating the structure of GAGs.

Chemical Synthesis of Fucosylated Chondroitin Sulfate Tetrasaccharide with Fucosyl Branch at the 6-OH of GalNAc Residue.

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D-glucuronic acid but, recently, a novel structure with α-L-fucose linked to the 6-OH of N-acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O-sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure-activity relationship in the future.

Cross-Reactive Polyclonal Antibodies Raised Against GalNAc-Conjugated siRNA Recognize Mostly the GalNAc Moiety.

Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.

Model-based meta-analysis to quantify the effects of short interfering RNA therapeutics on hepatitis B surface antigen turnover in hepatitis B-infected mice.

The objective of this study was to compare the efficacy of short interfering RNA therapeutics (siRNAs) in reducing hepatitis B surface antigen (HBsAg) levels in hepatitis B-infected (HBV) mice across multiple siRNA therapeutic classes using model-based meta-analysis (MBMA) techniques. Literature data from 10 studies in HBV-infected mice were pooled, including 13 siRNAs, formulated as liposomal nanoparticles (LNPs) or conjugated to either cholesterol (chol) or N-acetylgalactosamine (GalNAc). Time course of the baseline- and placebo-corrected mean HBsAg profiles were modeled using kinetics of drug effect (KPD) model coupled to an indirect response model (IRM) within a longitudinal non-linear mixed-effects MBMA framework. Single and multiple dose simulations were performed exploring the role of dosing regimens across evaluated siRNA classes. The HBsAg degradation rate (0.72 day-1) was consistent across siRNAs but exhibited a large between-study variability of 31.4% (CV%). The siRNA biophase half-life was dependent on the siRNA class and was highest for GalNAc-siRNAs (21.06 days) and lowest for chol-siRNAs (2.89 days). ID50 estimates were compound-specific and were lowest for chol-siRNAs and highest for GalNAc-siRNAs. Multiple dose simulations suggest GalNAc-siRNAs may require between 4 and 7 times less frequent dosing at higher absolute dose levels compared to LNP-siRNAs and chol-siRNAs, respectively, to reach equipotent HBsAg-lowering effects in HBV mice. In conclusion, non-clinical HBsAg concentration-time data after siRNA administration can be described using the presented KPD-IRM MBMA framework. This framework allows to quantitatively compare the effects of siRNAs on the HBsAg time course and inform dose and regimen selection across siRNA classes. These results may support siRNA development, optimize preclinical study designs, and inform data analysis methodology of future anti-HBV siRNAs; and ultimately, support siRNA model-informed drug development (MIDD) strategies.

Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain.

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.

Research progress on asialoglycoprotein receptor-targeted radiotracers designed for hepatic nuclear medicine imaging.

Asialoglycoprotein receptor (ASGPR) specifically recognizes glycans terminated with ß-d-galactose or N-acetylgalactosamine. Its exclusive expression in mammalian hepatocytes renders it an ideal hepatic-targeted biomarker. To date, ASGPR-targeted ligands have been actively developed for drug delivery and hepatic imaging. This review provides a comprehensive summary of the progress achieved to-date in the field of developing ASGPR-targeted nuclear medicine imaging (NMI) radiotracers, highlighting the recent advancements over the last decade in terms of structure, radionuclides and labeling strategies. The biodistribution patterns, imaging characteristics, challenges and future prospective are discussed.

RNA interference in the era of nucleic acid therapeutics.

Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N-acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today's RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions.

Deficiency of polypeptide N-acetylgalactosamine transferase 9 contributes to a risk for Parkinson's disease via mitochondrial dysfunctions.

Polypeptide N-acetylgalactosamine transferase 9 (GALNT9) catalyzes the initial step of mucin-type O-glycosylation via linking N-acetylgalactosamine (GalNAc) to serine/threonine in a protein. To unravel the association of GALNT9 with Parkinson's disease (PD), a progressive neurodegenerative disorder, GALNT9 levels were evaluated in the patients with PD and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and statistically analyzed based on the GEO datasets of GSE114918 and GSE216281. Glycoproteins with exposing GalNAc were purified using lectin affinity chromatography and identified by LC-MS/MS. The influence of GALNT9 on cells was evaluated via introducing a GALNT9-specific siRNA into SH-SY5Y cells. Consequently, GALNT9 deficiency was found to occur under PD conditions. GALNT9 silencing contributed to a causative factor in PD pathogenesis via reducing the levels of intracellular dopamine, tyrosine hydroxylase and soluble α-synuclein, and promoting α-synuclein aggregates. MS identification revealed 14 glycoproteins. 5 glycoproteins, including ACO2, ATP5B, CKB, CKMT1A, ALDOC, were associated with energy metabolism. GALNT9 silencing resulted in mitochondrial dysfunctions via increasing ROS accumulation, mitochondrial membrane depolarization, mPTPs opening, Ca2+ releasing and activation of the CytC-related apoptotic pathway. The dysfunctional mitochondria then triggered mitophagy, possibly intermediated by adenine nucleotide translocase 1. Our study suggests that GALNT9 is potentially developed into an auxiliary diagnostic index and therapeutic target of PD.

A combinatorial approach for achieving CNS-selective RNAi.

RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.

Safety and Tolerability of GalNAc3-Conjugated Antisense Drugs Compared to the Same-Sequence 2'-O-Methoxyethyl-Modified Antisense Drugs: Results from an Integrated Assessment of Phase 1 Clinical Trial Data.

The triantennary N-acetylgalactosamine (GalNAc3) cluster has demonstrated the utility of receptor-mediated uptake of ligand-conjugated antisense drugs targeting RNA expressed by hepatocytes. GalNAc3-conjugated 2'-O-methoxyethyl (2'MOE) modified antisense oligonucleotides (ASOs) have demonstrated a higher potency than the unconjugated form to support lower doses for an equivalent pharmacological effect. We utilized the Ionis integrated safety database to compare four GalNAc3-conjugated and four same-sequence unconjugated 2'MOE ASOs. This assessment evaluated data from eight randomized placebo-controlled dose-ranging phase 1 studies involving 195 healthy volunteers (79 GalNAc3 ASO, 24 placebo; 71 ASO, 21 placebo). No safety signals were identified by the incidence of abnormal threshold values in clinical laboratory tests for either ASO group. However, there was a significant increase in mean alanine transaminase levels compared with placebo in the upper dose range of the unconjugated 2'MOE ASO group. The mean percentage of subcutaneous injections leading to local cutaneous reaction was 30-fold lower in the GalNAc3-conjugated ASO group compared with the unconjugated ASO group (0.9% vs. 28.6%), with no incidence of flu-like reactions (0.0% vs. 0.7%). Three subjects (4.2%) in the unconjugated ASO group discontinued dosing. An improvement in the overall safety and tolerability profile of GalNAc3-conjugated 2'MOE ASOs is evident in this comparison of short-term clinical data in healthy volunteers.

Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

Application of improved GalNAc conjugation in development of cost-effective siRNA therapies targeting cardiovascular diseases.

N-Acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) therapies have received approval for treating both orphan and prevalent diseases. To improve in vivo efficacy and streamline the chemical synthesis process for efficient and cost-effective manufacturing, we conducted this study to identify better designs of GalNAc-siRNA conjugates for therapeutic development. Here, we present data on redesigned GalNAc-based ligands conjugated with siRNAs against angiopoietin-like 3 (ANGPTL3) and lipoprotein (a) (Lp(a)), two target molecules with the potential to address large unmet medical needs in atherosclerotic cardiovascular diseases. By attaching a novel pyran-derived scaffold to serial monovalent GalNAc units before solid-phase oligonucleotide synthesis, we achieved increased GalNAc-siRNA production efficiency with fewer synthesis steps compared to the standard triantennary GalNAc construct L96. The improved GalNAc-siRNA conjugates demonstrated equivalent or superior in vivo efficacy compared to triantennary GalNAc-conjugated siRNAs.

The deleterious variants of N-acetylgalactosamine-6-sulfatase (GalN6S) enzyme trigger Morquio a syndrome by disrupting protein foldings.

Lysosomal enzymes degrade cellular macromolecules, while their inactivation causes human hereditary metabolic disorders. Mucopolysaccharidosis IVA (MPS IVA; Moquio A syndrome) is one of the lysosomal storage disorders caused by a defective Galactosamine-6-sulfatase (GalN6S) enzyme. In several populations, disease incidence is elevated due to missense mutations brought on by non-synonymous allelic variation in the GalN6S enzyme. Here, we studied the effect of non-synonymous single nucleotide polymorphism (nsSNPs) on the structural dynamics of the GalN6S enzyme and its binding with N-acetylgalactosamine (GalNAc) using all-atom molecular dynamics simulation and an essential dynamics approach. Consequently, in this study, we have identified three functionally disruptive mutations in domain-I and domain-II, that is, S80L, R90W, and S162F, which presumably contribute to post-translational modifications. The study delineated that both domains work cooperatively, and alteration in domain II (S80L, R90W) leads to conformational changes in the catalytic site in domain-I, while mutation S162F mainly provokes higher residual flexibility of domain II. These results show that these mutations impair the hydrophobic core, implying that Morquio A syndrome is caused by misfolding of the GalN6S enzyme. The results also show the instability of the GalN6S-GalNAc complex upon substitution. Overall, the structural dynamics resulting from point mutations give the molecular rationale for Moquio A syndrome and, more importantly, the Mucopolysaccharidoses (MPS) family of diseases, re-establishing MPS IVA as a protein-folding disease.Communicated by Ramaswamy H. Sarma.

Gold nanoparticles decorated with monosaccharides and sulfated ligands as potential modulators of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS).

N-Acetylgalactosamine-6-sulfatase (GALNS) is an enzyme whose deficiency is related to the lysosomal storage disease Morquio A. For the development of effective therapeutic approaches against this disease, the design of suitable enzyme enhancers (i.e. pharmacological chaperones) is fundamental. The natural substrates of GALNS are the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate, which mainly display repeating units of sulfated carbohydrates. With a biomimetic approach, gold nanoparticles (AuNPs) decorated with simple monosaccharides, sulfated ligands (homoligand AuNPs), or both monosaccharides and sulfated ligands (mixed-ligand AuNPs) were designed here as multivalent inhibitors of GALNS. Among the homoligand AuNPs, the most effective inhibitors of GALNS activity are the ß-D-galactoside-coated AuNPs. In the case of mixed-ligand AuNPs, ß-D-galactosides/sulfated ligands do not show better inhibition than the ß-D-galactoside-coated AuNPs. However, a synergistic effect is observed for α-D-mannosides in a mixed-ligand coating with sulfated ligands that reduced IC50 by one order of magnitude with respect to the homoligand α-D-mannoside-coated AuNPs. SAXS experiments corroborated the association of GALNS with ß-D-galactoside AuNPs. These AuNPs are able to restore the enzyme activity by almost 2-fold after thermal denaturation, indicating a potential chaperoning activity towards GALNS. This information could be exploited for future development of nanomedicines for Morquio A. The recent implications of GALNS in cancer and neuropathic pain make these kinds of multivalent bionanomaterials of great interest towards multiple therapies.

N-acetylgalactosamine delivery systems for RNA therapeutics: a patent perspective.

INTRODUCTION: SiRNA molecules with a feature of good gene-silencing are critical for drug discovery and development based on RNA interference. GalNAc-RNA therapeutics is a rapid growing area in RNA therapeutics. AREAS COVERED: This article provides patent landscape and modification feature of GalNAc-RNA therapeutics. The US-granted patents from January 2004 to April 2023 were retrieved and analyzed. EXPERT OPINION: Globally, our study is the first one to holistically depict a map of modifications and therapeutic applications for GalNAc-RNA therapeutics by patent data analysis. The results showed there were 8 major modifications and 5 new emerged modifications for GalNAc-RNA therapeutic agents. Especially, the study provides recent new emerged modifications in sugar, base, and internucleotide linkage of GalNAc-RNA therapeutic agents, e.g. morpholino-type ring, 5-methylcytosine, and phosphorodithioates. In addition, our study systematically demonstrated major therapeutic applications for GalNAc-RNA therapeutics, including liver or gallbladder disorders, anticancer, antihyperlipidemics, and disorders of the nervous system etc.