Antisense therapy

Abstract: The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.

Abstract: For the majority of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has the potential to restore a functional protein. Here we show that weekly intravenous injections of morpholino phosphorodiamidate (morpholino) AONs induce expression of functional levels of dystrophin in body-wide skeletal muscles of the dystrophic mdx mouse, with resulting improvement in muscle function. Although the level of dystrophin expression achieved varies considerably between muscles, antisense therapy may provide a realistic hope for the treatment of a majority of individuals with DMD.

Abstract: Basic Principles of Antisense Technology Stanley T. Crooke Medicinal Chemistry of Antisense Oligonucleotides P. Dan Cook Analytical Methods for Antisense Drugs Janet M. Leeds and Lendell L. Cummins A Role for Antisense Technology in the Discovery of Highly Specific and Versatile Signal Transduction Inhibitors Brett P. Monia, Erich Koller, and William A. Gaarde Methods of Selecting Sites in RNA for Antisense Targeting Susan M. Freier Properties of Phosphorothioate Oligonucleotides Pharmacokinetic Properties in Animals Richard S. Geary, Rosie Z. Yu, Janet M. Leeds, Michael V. Templin, Tanya A. Watanabe, Scott P. Henry, and Arthur A. Levin Suborgan Pharmacokinetics Rosanne M. Crooke and Mark J. Graham Pharmacokinetic Properties in Humans Rosie Z. Yu, Steven L. Schoenfeld, Richard S. Geary, Tanya A. Watanabe, and Arthur A. Levin Toxicity of Antisense Oligonucleotides Arthur A. Levin, Scott P. Henry, David Monteith, and Michael V. Templin Clinical Safety of Phosphorothioate Oligodeoxynucleotides F. Andrew Dorr, Josephine M. Glover, and T. Jesse Kwoh General Pharmacology of Phosphorothioate Oligodeoxynucleotides C. Frank Bennett Properties of Advanced Novel Clinical Classes of Oligonucleotides Pharmacology of 2'-0-(2-Methoxy)ethyl Modified Antisense Oligonucleotides Nicholas M. Dean, Madeline Butler, Brett P. Monia, and Muthiah Manoharan Locked Nucleic Acid Jesper Wengel Antisense Properties of Peptide Nucleic Acid (PNA) Uffe Koppelhus and Peter J. Nielsen Phosphorodiamidate Morpholino Oligomers Patrick Iversen Oligonucleotide Conjugates in Antisense Technology Muthiah Manoharan Immune Stimulation by Oligonucleotides Arthur M. Krieg Pre-mRNA Splicing as a Target for Antisense Oligonucleotides Ryszard Kole and Danielle Mercatante Application of Antisense Oligonucleotides to the Study of CNS Protein Function Siew Peng Ho Antisense Approach to Isoform-Specific Blockade of Acetylcholinesterase Hermona E. Soreq and Shlomo Seidman Serine/Threonine Protein Phosphates Richard E. Honkanen Pharmacological Activities of Antisense Drugs: Inflammatory Diseases James G. Karras, Kathleen J. Myers, and Brenda F. Baker Respirable Antisense Oligonucleotides (RASONs) Jonathan W. Nyce Combined Antisense Therapy and Chemotherapy in Animal Models Dan Mercola Antisense Oligodesoxynucleotide Strategies in Renal and Cardiovascular Disease Hermann Haller, Christian Maasch, Duska Dragun, Maren Wellner, and Friedrich C. Luft The Development of Antisense Oligonucleotides as Antivirals Lisa R. Grillone Clinical Activities in Patients with Solid Tumors or Lymphoma Jon T. Holmlund Nucleic Acid Therapeutics for the Treatment of Human Leukemia Alan M. Gewirtz ISIS 2302, an Antisense Inhibitor of Intercellular Adhesion Molecule 1 (ICAM-1) William R. Shanahan, Jr. New Routes and Novel Formulations for Delivery of Antisense Oligonucleotides Gregory E. Hardee, Susan P. Weinbach, and Lloyd G. Tillman DNA-Binding Molecules Roland W. Burli and Heinz E. Moser Targeted Genome Modification via Triple Helix Formation Margaret A. Macris and Peter M. Glazer Intracellular Ribozyme Applications John J. Rossi