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Journal of the American Chemical Society Dec 2014Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the...
Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.
Topics: Acetylgalactosamine; Animals; Gene Silencing; Hepatocytes; Mice; Mice, Inbred C57BL; Molecular Structure; RNA, Small Interfering
PubMed: 25434769
DOI: 10.1021/ja505986a -
The Journal of Pharmacology and... Nov 2021Conjugation of small interfering RNA (siRNA) to tris -acetylgalactosamine [(GalNAc)] can enable highly selective, potent, and durable knockdown of targeted proteins in...
Minimal Physiologically Based Pharmacokinetic-Pharmacodynamic (mPBPK-PD) Model of -Acetylgalactosamine-Conjugated Small Interfering RNA Disposition and Gene Silencing in Preclinical Species and Humans.
Conjugation of small interfering RNA (siRNA) to tris -acetylgalactosamine [(GalNAc)] can enable highly selective, potent, and durable knockdown of targeted proteins in the liver. However, potential knowledge gaps between in vitro experiments, preclinical species, and clinical scenarios remain. A minimal physiologically based pharmacokinetic-pharmacodynamic model for GalNAc-conjugated siRNA (GalNAc-siRNA) was developed using published data for fitusiran (ALN-AT3), an investigational compound targeting liver antithrombin (AT), to delineate putative determinants governing the whole-body-to-cellular pharmacokinetic (PK) and pharmacodynamic (PD) properties of GalNAc-siRNA and facilitate preclinical-to-clinical translation. The model mathematically linked relevant mechanisms: 1) hepatic biodistribution, 2) tris-GalNAc binding to asialoglycoprotein receptors (ASGPRs) on hepatocytes, 3) ASGPR endocytosis and recycling, 4) endosomal transport and escape of siRNA, 5) cytoplasmic RNA-induced silencing complex (RISC) loading, 6) degradation of target mRNA by bound RISC, and 7) knockdown of protein. Physiologic values for 36 out of 48 model parameters were obtained from the literature. Kinetic parameters governing (GalNAc)-ASGPR binding and internalization were derived from published studies of uptake in hepatocytes. The proposed model well characterized reported pharmacokinetics, RISC dynamics, and knockdown of AT mRNA and protein by ALN-AT3 in mice. The model bridged multiple PK-PD data sets in preclinical species (mice, rat, monkey) and successfully captured reported plasma pharmacokinetics and AT knockdown in a phase I ascending-dose study. Estimates of in vivo potency were similar (∼2-fold) across species. Subcutaneous absorption and serum AT degradation rate constants scaled across species by body weight with allometric exponents of -0.29 and -0.22. The proposed mechanistic modeling framework characterizes the unique PK-PD properties of GalNAc-siRNA. SIGNIFICANCE STATEMENT: Tris -acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) therapeutics enable liver-targeted gene therapy and precision medicine. Using a translational and systems-based minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) modeling approach, putative determinants influencing GalNAc-conjugated siRNA (GalNAc-siRNA) functionality in three preclinical species and humans were investigated. The developed model successfully integrated and characterized relevant published in vitro-derived biomeasures, mechanistic PK-PD profiles in animals, and observed clinical PK-PD responses for an investigational GalNAc-siRNA (fitusiran). This modeling effort delineates the disposition and liver-targeted pharmacodynamics of GalNAc-siRNA.
Topics: Acetylgalactosamine; Animals; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Gene Silencing; Haplorhini; Hepatocytes; Humans; Mice; Models, Biological; RNA, Small Interfering; Rats; Tissue Distribution
PubMed: 34413198
DOI: 10.1124/jpet.121.000805 -
Signal Transduction and Targeted Therapy Jun 2020RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a... (Review)
Review
RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality. After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO (patisiran) and GIVLAARI™ (givosiran), have been achieved by Alnylam Pharmaceuticals. Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases. It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies. The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects. In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed. All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)-siRNA conjugate, and their fundamental design principles are thoroughly discussed. The latest progress in siRNA therapeutic development is also summarized. This review provides a comprehensive view and roadmap for general readers working in the field.
Topics: Acetylgalactosamine; Genetic Diseases, Inborn; Genetic Therapy; Humans; Pyrrolidines; RNA Interference; RNA, Double-Stranded; RNA, Small Interfering
PubMed: 32561705
DOI: 10.1038/s41392-020-0207-x -
Nature Nanotechnology Jun 2021The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional... (Review)
Review
The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. Their clinical translation, however, depends on delivery technologies that improve stability, facilitate internalization and increase target affinity. Here, we review four platform technologies that have enabled the clinical translation of nucleic acid therapeutics: antisense oligonucleotides, ligand-modified small interfering RNA conjugates, lipid nanoparticles and adeno-associated virus vectors. For each platform, we discuss the current state-of-the-art clinical approaches, explain the rationale behind its development, highlight technological aspects that facilitated clinical translation and provide an example of a clinically relevant genetic drug. In addition, we discuss how these technologies enable the development of cutting-edge genetic drugs, such as tissue-specific nucleic acid bioconjugates, messenger RNA and gene-editing therapeutics.
Topics: Acetylgalactosamine; Gene Editing; Gene Expression Regulation; Genetic Vectors; Humans; Lipids; Nanoparticles; Nucleic Acids; Oligonucleotides; Oligonucleotides, Antisense; Pyrrolidines; RNA, Small Interfering
PubMed: 34059811
DOI: 10.1038/s41565-021-00898-0 -
Nature Chemical Biology Sep 2021Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting...
Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.
Topics: Acetylgalactosamine; Asialoglycoprotein Receptor; Humans; Lysosomes; Tumor Cells, Cultured
PubMed: 33767387
DOI: 10.1038/s41589-021-00770-1 -
Journal of Chromatography. A Jan 2022N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing...
N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.5 × 300 mm, 13 µm), with the former affording double the recovery and higher purity of 95% over its AEX counterpart displaying 91% for a selected siRNA conjugate. An assortment of GalNAc-modified oligonucleotides was later purified using the mixed-mode resin revealing good recoveries (∼30-60%) and high purities of 90-94% ranging from straightforward to more challenging purifications. High sample loading in the 20 mg range was achieved, which was comparable with the larger preparative TSKgel SuperQ-5PW support. The Scherzo-SS-C18 resin also afforded some degree of resolution between diastereomers containing phosphorothioate functionalities. The TSKgel SuperQ-5PW support was later investigated to provide orthogonal separation selectivity to the Scherzo-SS-C18 column enabling purification of a selected, GalNAc-siRNA conjugate. The developed pH (8.5-11) and salt (0.3-0.7 M) gradients method provided enhanced separation selectivity between the free and conjugated siRNA, while minimizing formation of secondary structures and highlighting a complementary approach to deal with challenging purifications of oligonucleotide-GalNAc conjugates. Together, the use of AEX and mixed-mode columns provide much needed orthogonality to deal with complex GalNAc-modified oligonucleotides and potentially other upcoming modalities.
Topics: Acetylgalactosamine; Anions; Chromatography, Ion Exchange; Oligonucleotides
PubMed: 34871941
DOI: 10.1016/j.chroma.2021.462679 -
The Journal of Physical Chemistry. A Oct 2022In the present work, we report the first rotational study of -acetylgalactosamine, a cancer-associated sugar derivative, by means of high-resolution rotational...
In the present work, we report the first rotational study of -acetylgalactosamine, a cancer-associated sugar derivative, by means of high-resolution rotational spectroscopy. Two different conformers have been conclusively characterized using broadband Fourier transform microwave spectroscopy coupled with a laser ablation vaporization system. Additionally, we performed a comprehensive analysis of the intramolecular interactions that govern these structures, which allowed us to both characterize the existence of intramolecular hydrogen bond networks that drive the intrinsic conformation panorama of -acetylgalactosamine and further rationalize the biological role of this aminosugar derivative as part of the Tn antigen.
Topics: Humans; Acetylgalactosamine; Molecular Conformation; Hydrogen Bonding; Organic Chemicals; Neoplasms; Sugars
PubMed: 36099577
DOI: 10.1021/acs.jpca.2c04595 -
Nature Biomedical Engineering Oct 2019Homeostatic antigen presentation by hepatic antigen-presenting cells, which results in tolerogenic T-cell education, could be exploited to induce antigen-specific...
Homeostatic antigen presentation by hepatic antigen-presenting cells, which results in tolerogenic T-cell education, could be exploited to induce antigen-specific immunological tolerance. Here we show that antigens modified with polymeric forms of either N-acetylgalactosamine or N-acetylglucosamine target hepatic antigen-presenting cells, increase their antigen presentation and induce antigen-specific tolerance, as indicated by CD4 and CD8 T-cell deletion and anergy. These synthetically glycosylated antigens also expanded functional regulatory T cells, which are necessary for the durable suppression of antigen-specific immune responses. In an adoptive-transfer mouse model of type-1 diabetes, treatment with the glycosylated autoantigens prevented T-cell-mediated diabetes, expanded antigen-specific regulatory T cells and resulted in lasting tolerance to a subsequent challenge with activated diabetogenic T cells. Glycosylated autoantigens targeted to hepatic antigen-presenting cells might enable therapies that promote immune tolerance in patients with autoimmune diseases.
Topics: Acetylgalactosamine; Acetylglucosamine; Adoptive Transfer; Animals; Antigen Presentation; Autoantigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Immune Tolerance; Liver; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Spleen; T-Lymphocytes
PubMed: 31358881
DOI: 10.1038/s41551-019-0424-1 -
The New England Journal of Medicine Jan 2017
Topics: Acetylgalactosamine; Anticholesteremic Agents; Drug Discovery; Hepatocytes; Humans; RNA, Small Interfering; RNAi Therapeutics
PubMed: 28052224
DOI: 10.1056/NEJMp1614154 -
Nature Communications Feb 2018Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of...
Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.
Topics: Acetylgalactosamine; Animals; Liver; Male; RNA Interference; RNA, Small Interfering; Rats; Rats, Sprague-Dawley
PubMed: 29459660
DOI: 10.1038/s41467-018-02989-4