Glycosylation in Cellular Mechanisms of Health and Disease

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

The discovery that gene expression can be controlled by the Watson–Crick base-pairing of small RNAs with messenger RNAs containing complementary sequence — a process known as RNA interference — has markedly advanced our understanding of eukaryotic gene regulation and function. The ability of short RNA sequences to modulate gene expression has provided a powerful tool with which to study gene function and is set to revolutionize the treatment of disease. Remarkably, despite being just one decade from its discovery, the phenomenon is already being used therapeutically in human clinical trials, and biotechnology companies that focus on RNA-interference-based therapeutics are already publicly traded.

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The promises and pitfalls of RNA-interference-based therapeutics

Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard Thomas C. Terwilliger a* , Ralf W. Grosse-Kunstleve b , Pavel V. Afonine b , Nigel W. Moriarty b , Peter Zwart b , Li-Wei Hung a , Randy J. Read c , Paul D. Adams b* a b Los Alamos National Laboratory, Mailstop M888, Los Alamos, NM 87545, USA Lawrence Berkeley National Laboratory, One Cyclotron Road, Bldg 64R0121, Berkeley, CA 94720, USA. c Department of Haematology, University of Cambridge, Cambridge CB2 0XY, UK. * Email: terwill@lanl.gov or PDAdams@lbl.gov Running title: The PHENIX AutoBuild Wizard Abstract The PHENIX AutoBuild Wizard is a highly automated tool for iterative model- building, structure refinement and density modification using RESOLVE or TEXTAL model- building, RESOLVE statistical density modification, and phenix.refine structure refinement. Recent advances in the AutoBuild Wizard and phenix.refine include automated detection and application of NCS from models as they are built, extensive model completion algorithms, and automated solvent molecule picking. Model completion algorithms in the AutoBuild Wizard include loop-building, crossovers between chains in different models of a structure, and side-chain optimization. The AutoBuild Wizard has been applied to a set of 48 structures at resolutions ranging from 1.1 A to 3.2 A, resulting in a mean R-factor of 0.24 and a mean free R factor of 0.29. The R-factor of the final model is dependent on the quality of the starting electron density, and relatively independent of resolution. Keywords: Model building; model completion; macromolecular models; Protein Data Bank; structure refinement; PHENIX Introduction Iterative model-building and refinement is a powerful approach to obtaining a complete and accurate macromolecular model. The approach consists of cycles of building an atomic model based on an electron density map for a macromolecular structure, refining the structure, using the refined structure as a basis for improving the map, and building a new model. This type of approach has been carried out in a semi-automated fashion for many years, with manual model-building iterating with automated refinement (Jensen, 1997). More recently, with the development first of ARP/wARP (Perrakis et al., 1999), and later other procedures including RESOLVE iterative model-building and refinement (Terwilliger,

Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.

Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells.

The Allyloxycarbonyl (Aloc) Moiety—Conversion of an Unsuitable into a Valuable Amino Protecting Group for Peptide Synthesis†

Incremental developments in the chemistry of peptides, proteins and carbohydrates have enabled researchers to assemble entire glycoproteins with high precision. Based on sophisticated ligation chemistries pure glycoproteins bearing a single glycosylation pattern have become available. The impact of N-glycosylation on the function of glycoproteins is generally recognized but not well understood. Based on the recent advances in the synthesis of glycoproteins by chemical methods researchers can finally start to elucidate the various roles of carbohydrates in complex biomolecules in detail.

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Chemical assembly of N-glycoproteins: a refined toolbox to address a ubiquitous posttranslational modification

High-efficiency synthesis of sialyloligosaccharides and sialoglycopeptides

Endogenous ligands have not, to date, been identified for the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed by parenchymal cells in the liver of mammals. On the basis of the rapid clearance of BSA bearing multiple chemically coupled sialic acid (Sia)α2,6GalNAcβ1,4GlcNAcβ1,2Man tetrasaccharides (SiaGGnM-BSA) from the circulation, and the ability of the ASGP-R hepatic lectin-1 subunit to bind SiaGGnM-BSA, we previously proposed that glycoproteins modified with structures terminating with Siaα2,6GalNAc may represent previously unrecognized examples of endogenous ligands for this receptor. Here, we have taken a genetic approach using wild-type and ASGP-R-deficient mice to determine that the ASGP-R in vivo does indeed account for the rapid clearance of glycoconjugates terminating with Siaα2,6GalNAc. We have also determined that the ASGP-R is able to bind core-substituted oligosaccharides with the terminal sequence Siaα2,6Galβ1,4GlcNAc but not those with the terminal Siaα2,3Galβ1,4GlcNAc. We propose that glycoproteins bearing terminals Siaα2,6GalNAc and Siaα2,6Gal are endogenous ligands for the ASGP-R, and that the ASGP-R helps to regulate the relative concentration of serum glycoproteins bearing α2,6-linked Sia.

https://www.pnas.org/content/pnas/102/47/17125.full.pdf

Carlo Unverzagt

Papers

Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells.

The Allyloxycarbonyl (Aloc) Moiety—Conversion of an Unsuitable into a Valuable Amino Protecting Group for Peptide Synthesis†

Chemical assembly of N-glycoproteins: a refined toolbox to address a ubiquitous posttranslational modification

High-efficiency synthesis of sialyloligosaccharides and sialoglycopeptides

The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acidα2,6GalNAc