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Rudolph L. Juliano - One of the best experts on this subject based on the ideXlab platform.
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The delivery of therapeutic Oligonucleotides.
Nucleic Acids Research, 2016Co-Authors: Rudolph L. JulianoAbstract:: The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching Oligonucleotides. However, effective delivery of Oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of Oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of Oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology.
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cellular uptake and intracellular trafficking of Oligonucleotides
Advanced Drug Delivery Reviews, 2015Co-Authors: Rudolph L. Juliano, Kyle CarverAbstract:Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how Oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and intracellular trafficking of both ‘free’ Oligonucleotides and Oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand oligonucleotide delivery. In particular we discuss opportunities for escape of Oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance oligonucleotide effects.
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cellular uptake and intracellular trafficking of Oligonucleotides implications for oligonucleotide pharmacology
Nucleic Acid Therapeutics, 2014Co-Authors: Rudolph L. Juliano, Kyle Carver, Xin Ming, Brian LaingAbstract:One of the major constraints on the therapeutic use of Oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of Oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of Oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed.
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Conjugates of antisense Oligonucleotides with the Tat and Antennapedia cell-penetrating peptides: Effects on cellular uptake, binding to target sequences, and biologic actions
Pharmaceutical Research, 2002Co-Authors: Anna Astriab-fisher, Dimitri Sergueev, Barbara Ramsay Shaw, Michael Fisher, Rudolph L. JulianoAbstract:PURPOSE: The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense Oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2'-O-methyl phosphorothioate Oligonucleotides. METHODS: The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques. RESULTS: The peptide-oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the Oligonucleotides were also examined using surface plasmon resonance. The peptide-oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated Oligonucleotides. CONCLUSIONS: Conjugation with cell-penetrating peptides enhances oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense Oligonucleotides.
Ramon Eritja - One of the best experts on this subject based on the ideXlab platform.
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Biophysical and RNA Interference Inhibitory Properties of Oligonucleotides Carrying Tetrathiafulvalene Groups at Terminal Positions
Journal of Chemistry, 2013Co-Authors: Sónia Pérez-rentero, Álvaro Somoza, Jiří Janoušek, Martin Bělohradský, Santiago Grijalvo, Irena G. Stará, Ivo Starý, Ramon EritjaAbstract:Oligonucleotide conjugates carrying a single functionalized tetrathiafulvalene (TTF) unit linked through a threoninol molecule to the 3′ or 5′ ends were synthesized together with their complementary Oligonucleotides carrying a TTF, pyrene, or pentafluorophenyl group. TTF-oligonucleotide conjugates formed duplexes with higher thermal stability than the corresponding unmodified Oligonucleotides and pyrene- and pentafluorophenyl-modified Oligonucleotides. TTF-modified Oligonucleotides are able to bind to citrate-stabilized gold nanoparticles (AuNPs) and produce stable gold AuNPs functionalized with Oligonucleotides. Finally, TTF-oligoribonucleotides have been synthesized to produce siRNA duplexes carrying TTF units. The presence of the TTF molecule is compatible with the RNA interference mechanism for gene inhibition.
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Stepwise synthesis of oligonucleotide–peptide conjugates containing guanidinium and lipophilic groups in their 3′-termini
Bioorganic & Medicinal Chemistry Letters, 2010Co-Authors: Santiago Grijalvo, Montserrat Terrazas, Anna Aviñó, Ramon EritjaAbstract:Abstract Two different series of oligonucleotide–peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, Oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide–phosphorothioate conjugates were evaluated as potential antisense Oligonucleotides in the inhibition of the luciferase gene.
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Solid-Phase Synthesis of Modified Oligonucleotides
International Journal of Peptide Research and Therapeutics, 2007Co-Authors: Ramon EritjaAbstract:Synthetic Oligonucleotides are ubiquitously found in most laboratories since solid-phase synthesis protocols have become highly optimized. These protocols make it possible to synthesize a large variety of modified Oligonucleotides. As one example, we will review some of the developments regarding oligonucleotide synthesis from our own group. In particular, we will describe the synthesis of Oligonucleotides carrying non-natural bases, of oligonucleotide–peptide conjugates, and of modified Oligonucleotides used in the assembly of nanomaterials.
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Digoxigenin-Labeled Phosphorothioate Oligonucleotides: A New Tool for the Study of Cellular Uptake
Antisense research and development, 1995Co-Authors: Gemma Tarrason, David Bellido, Senén Vilaró, Ramon Eritja, Jaume PiulatsAbstract:The mechanisms and intracellular pathways by which many oligonucleotide analogs enter cells to exert the desired antisense effects are not fully understood and remain a matter of debate. In this study, we describe the synthesis of 5′-digoxigenin-labeled phosphorothioate Oligonucleotides and show their use to examine intracellular oligonucleotide distribution within Epstein-Harr virus-transformed B cells. Comparison of digoxigeninlabeled and fluorescein-labeled oligonucleotide distribution shows the same intracellular fate, suggesting that digoxigenin modification does not interfere with intracellular routing. Double immunofluorescence studied by conventional fluorescence and confocal microscopy with antibodies to the labeling molecule and to lysosomeassociated membrane protein indicate that Oligonucleotides mainly accumulate in the lysosomal compartment. Digoxigenin labeling offers an alternative to study oligonucleotide uptake and distribution by immunoelectron microscopy. Two different approaches have b...
Kyle Carver - One of the best experts on this subject based on the ideXlab platform.
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cellular uptake and intracellular trafficking of Oligonucleotides
Advanced Drug Delivery Reviews, 2015Co-Authors: Rudolph L. Juliano, Kyle CarverAbstract:Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how Oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and intracellular trafficking of both ‘free’ Oligonucleotides and Oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand oligonucleotide delivery. In particular we discuss opportunities for escape of Oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance oligonucleotide effects.
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cellular uptake and intracellular trafficking of Oligonucleotides implications for oligonucleotide pharmacology
Nucleic Acid Therapeutics, 2014Co-Authors: Rudolph L. Juliano, Kyle Carver, Xin Ming, Brian LaingAbstract:One of the major constraints on the therapeutic use of Oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of Oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of Oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed.
Sudhir Agrawal - One of the best experts on this subject based on the ideXlab platform.
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Antisense and/or immunostimulatory oligonucleotide therapeutics.
Current Cancer Drug Targets, 2001Co-Authors: Sudhir Agrawal, Kandimalla ErAbstract:: Antisense technology, which is based on a simple and rational principle of Watson-Crick complementary base pairing of a short oligonucleotide with the targeted mRNA to downregulate the disease-causing gene product, has progressed tremendously in the last two decades. Antisense Oligonucleotides targeted to a number of cancer-causing genes are being evaluated in human clinical trials. While the first-generation phosphorothioate antisense Oligonucleotides are in clinical trials, a number of factors, including sequence motifs that could lead to unwanted mechanisms of action and side effects, have been identified. The severity of the side effects of first-generation antisense Oligonucleotides is mostly dependent on the presence of certain sequence motifs, such as CpG dinucleotides. A number of second-generation chemical modifications have been proposed to overcome the limitations of the first-generation antisense Oligonucleotides. The safety and efficacy of several second-generation mixed-backbone antisense Oligonucleotides are being evaluated in clinical trials. The immune stimulation affects observed with CpG-containing antisense Oligonucleotides are being exploited as a novel therapeutic modality, with several CpG Oligonucleotides being evaluated in clinical trials. A number of medicinal chemistry studies performed to date suggest that the immunomodulatory activity of CpG Oligonucleotides can be fine-tuned by site-specific incorporation of chemical modifications in order to design disease-specific oligonucleotide therapeutics.
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Effect of aspirin on protein binding and tissue disposition of oligonucleotide phosphorothioate in rats
Journal of Drug Targeting, 1998Co-Authors: Sudhir Agrawal, Xueshu Zhang, Qiuyin Cai, Ekambar R. Kandimalla, Alisa K. Manning, Zhiwie Jiang, T. Marcel, Ruiwen ZhangAbstract:AbstractPharmacokinetic studies of phosphorothioate oligodeoxynucleotides (PS-Oligonucleotides) in animals show that following intravenous administration, PS-oligonucleotide clears out rapidly from the plasma and is distributed to majority of the organs. PS-Oligonucleotides are bound to plasma proteins extensively. This study was aimed to determine the effect of aspirin, a commonly used drug, on pharmacokinetics of PS-Oligonucleotides. In the present study, PS-oligonucleotide was administered to rats that had received aspirin by gavage. Pharmacokinetic study shows that if PS-oligonucleotide was administered following aspirin administration in rats, a) plasma pharmacokinetic parameters (t1/2α?, t1/2β, AUC, etc.) had lower values, b) tissue disposition was different, and c) rate and route of elimination was affected in animals compared to rats receiving PS-oligonucleotide alone. This finding suggests that pharmacokinetics of PS-Oligonucleotides can be affected with certain class of drugs, which may have dir...
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Effect of different chemically modified oligodeoxynucleotides on immune stimulation
Biochemical Pharmacology, 1996Co-Authors: Qiuyan Zhao, Jamal Temsamani, Zhiwei Jiang, Patricia L. Iadarola, Sudhir AgrawalAbstract:Based on previous studies that certain Oligonucleotides can stimulate cell proliferation and immunoglobulin production, this study was carried out to establish the relationship between the stimulatory effect and the chemical modification of the oligonucleotide. First, the effects of oligonucleotide and analogs on immune stimulation were studied in vitro using murine splenic lymphocytes. Our results show that cell proliferation and immunoglobulin production (IgG and IgM) depend on the sequence and the chemical modification of the oligonucleotide. Phosphorothioate oligodeoxynucleotides displayed a greater stimulatory effect than partially modified phosphorothioate Oligonucleotides. Second, we studied the effects of these chemically modified olionucleotides after injection in mice. Massive splenomegaly and stimulation of cell proliferation were observed with some phosphorothioate Oligonucleotides. These effect were minimized markedly by chimeric and hybrid Oligonucleotides. We also demonstrate that in vitro the effects of Oligonucleotides on murine lymphocytes were unaffected by T cell depletion, suggesting that Oligonucleotides exert their effects mainly on the B cells.
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Pharmacokinetics of Antisense Oligonucleotides
Clinical Pharmacokinetics, 1995Co-Authors: Sudhir Agrawal, Jamal Temsamani, Wayne Galbraith, Jinyan TangAbstract:Antisense Oligonucleotides are promising therapeutic agents for the treatment of life-threatening diseases. Intravenous injection of phosphodiester oligonucleotide analogue (P-oligonucleotide) in monkeys shows that the oligonucleotide is degraded rapidly in the plasma with a half-life of about 5 minutes. Administration of a single dose of the phosphorothioate (S-oligonucleotide) in animals by the intravenous route reveals biphasic plasma elimination. An initial short half-life (0.53 to 0.83 hours) represents distribution out of the plasma compartment and a second long half-life (35 to 50 hours) represents elimination from the body. This elimination half-life was similar when the oligonucleotide was administered subcutaneously. In contrast, methylphosphonate Oligonucleotides have an elimination half-life of 17 minutes in mice. S-Oligonucleotide was distributed into most of organs of rats and mice. Liver and kidney were the 2 organs with highest uptake of the oligonucleotide. The S-oligonucleotide was primarily excreted in urine. Up to 30% was excreted in the first 24 hours. Repeated daily intravenous injections of a 25-mer S-oligonucleotide into rats showed that the concentrations in the plasma are at steady-state during the 8 days’ administration. The data represented here support the potential utility of phosphorothioate and methylphosphonate Oligonucleotides as therapeutic agents in vivo .
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Pharmacokinetics, biodistribution, and stability of capped oligodeoxynucleotide phosphorothioates in mice.
Antisense Research and Development, 1993Co-Authors: Jamal Temsamani, Jinyan Tang, Abeysinghe Padmapriya, Michael Kubert, Sudhir AgrawalAbstract:Several end-modified oligodeoxynucleotide phosphorothioates (S-Oligonucleotides) were studied for their pharmacokinetics, biodistribution, excretion, and metabolic stability in vivo after intravenous administration in mice. The overall tissue distribution and excretion patterns of these S-Oligonucleotides were found to be independent of 5' or 3' end modification studied. However, the 3' end modification proved to be of considerable importance with respect to metabolic stability of the oligonucleotide. In the case of uncapped and 5'-capped S-oligonucleotide, only 50% of intact oligonucleotide was recovered out of the total bioavailable concentration in liver and kidney. In contrast, in the case of 3'-capped Oligonucleotides almost all bioavailable concentrations of 3'-capped oligonucleotide was found to be intact in kidney and liver at 24 hr after administration. These results demonstrate that superior pharmaceutical potential can be created by 3'-end modification of oligonucleotide phosphorothioates.
Yogesh S. Sanghvi - One of the best experts on this subject based on the ideXlab platform.
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Tandem oligonucleotide synthesis on solid-phase supports for the production of multiple Oligonucleotides.
Journal of Organic Chemistry, 2002Co-Authors: Shuyuan Yu, Yogesh S. SanghviAbstract:More than one oligonucleotide can be synthesized at a time by linking multiple Oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5‘-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two Oligonucleotides are linked via a cleavable 3‘-O-hydroquinone-O,O‘-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5‘-OH position of immobilized Oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3‘- or 5‘-end of Oligonucleotides. A single NH4OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5‘- and 3‘-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucle...