Open Access Journal of Drug Discovery, Development and Delivery Special Article - Antisense Drug Research and Development Guide for Morpholino Users: Toward Therapeutics Moulton JD* Gene Tools, LLC, USA Abstract *Corresponding author: Moulton JD, Gene Tools, Morpholino oligos are uncharged molecules for blocking sites on RNA. They LLC, 1001 Summerton Way, Philomath, Oregon 97370, are specific, soluble, non-toxic, stable, and effective antisense reagents suitable USA for development as therapeutics and currently in clinical trials. They are very versatile, targeting a wide range of RNA targets for outcomes such as blocking Received: January 28, 2016; Accepted: April 29, 2016; translation, modifying splicing of pre-mRNA, inhibiting miRNA maturation and Published: May 03, 2016 activity, as well as less common biological targets and diagnostic applications. Solutions have been developed for delivery into a range of cultured cells, embryos and adult animals; with development of a non-toxic and effective system for systemic delivery, Morpholinos have potential for broad therapeutic development targeting pathogens and genetic disorders. Keywords: Splicing; Duchenne muscular dystrophy; Phosphorodiamidate morpholino oligos; Internal ribosome entry site; Nonsense-mediated decay Morpholinos: Research Applications, the transcript from miRNA regulation; Therapeutic Promise • Block regulatory proteins from binding to RNA, shifting Morpholino oligos bind to complementary sequences of RNA alternative splicing; and get in the way of processes. Morpholino oligos are commonly • Block association of RNAs with cytoskeletal motor protein used to prevent a particular protein from being made in an organism complexes, preventing RNA translocation; or cell culture. Morpholinos are not the only tool used for this: a protein’s synthesis can be inhibited by altering DNA to make a null • Inhibit poly-A tailing of pre-mRNA; mutant (called a gene knockout) or by interrupting processes on RNA • Trigger frame shifts at slippery sequences; (called a gene knockdown). Some DNA alterations cause production of a protein to decrease without stopping all production; confusingly, • Invade RNA secondary structure, changing its these are also called gene knockdowns. DNA alterations are conformation; permanent, while knockdowns of RNA are either transient, generally • Serve as guide sequences for RNase-P activity; or last several days after dosing with antisense (such as Morpholinos), or are long-term, depending on continued production of knockdown • Block activity of a ribozyme. RNA in cells (such as shRNA transcribed in cells from a plasmid). Morpholinos have been broadly used in the developmental biology Once introduced into cells, Morpholinos freely diffuse between community to knock down genes in embryos of organisms such as the cytosol and nuclear compartments and bind complementary zebrafish (Danio rerio), African clawed frogs (Xenopus sp.), chicks sequences of RNA. Morpholinos have been used as gene knockdown (Gallus gallus), sea urchins (e.g. Strongylocentrotus sp.), sea squirts reagents in cell cultures and in animals, bacteria, protists, plants and (Ciona sp.), and many more. The oligos are usually microinjected fungi. The RNA-blocking property of Morpholinos has also been through fine glass needles into early embryos at the one-to-few used to cause a range of different outcomes beyond simple gene cell stage. Many kinds of antisense have toxic effects during knockdowns. Morpholinos have different effects depending on the development of an embryo. Because Morpholinos have little sort of target they bind. Morpholinos can: interaction with protein they are unusually non-toxic antisense, sufficiently non-toxic to make them the first choice of most • Block ribosome assembly and stop translation of a protein developmental biologists for transient gene knockdowns. In from an mRNA; contrast, injection of oligos containing phosophorothioate • Bind splice junctions and deny access to the small intersubunit linkages often kills embryos [1]. Morpholinos are nuclear Ribonuclear Proteins (snRNPs) that mark the junctions for highly specific antisense, having less interaction with unintended spliceosomes, altering splicing; RNAs than antisense which employs protein activity; this is because a Morpholino must be complementary to a longer • Bind to precursors of miRNA, inhibiting the maturation of sequence of RNA than antisense using catalytic activity (e.g. RNAi, the miRNA; phosphorothioate DNA, etc.) [2]. Less specific antisense causes • Bind to mature miRNA, inhibiting the activity of the changes in gene expression during development of the embryo and miRNA; can cause developmental defects (teratogenesis) [3]. • Bind to miRNA recognition elements on mRNA, relieving If you want to use an antisense oligo for a therapeutic, non- toxicity and specificity are excellent characteristics with which to J Drug Discov Develop and Deliv - Volume 3 Issue 2 - 2016 Citation: Moulton JD. Guide for Morpholino Users: Toward Therapeutics. J Drug Discov Develop and Deliv. ISSN : 2471-0288 | www.austinpublishinggroup.com 2016; 3(2): 1023. Moulton. © All rights are reserved Moulton JD Austin Publishing Group start. Morpholinos are also reasonably water soluble, with most oligos is referred to as RNase-H independent antisense. Some other oligo staying in solution for many months to years at 1mM concentration types, such as 2’-O-methyl phosphorothioate, peptide nucleic acid, in room temperature water; this gives them a great advantage over and locked nucleic acid oligos, are also steric blocking, RNase-H another kind of potential therapeutic antisense, the Peptide Nucleic independent antisense. Acids (PNA) [4]. Clinical trials conducted by Sarepta Theraeputics Benefits of Morpholino Structure Inc. (formerly AVI BioPharma Inc.) have shown excellent safety of the oligos in humans (Table 1). Lack of electrostatic charge Efficacy of Morpholino oligos in humans has been shown We hypothesize that the lack of protein interaction with in clinical trials for Duchenne muscular dystrophy. The splice- Morpholinos is due to their lack of electrostatic charge. Nucleic acids interact with proteins through relatively weak hydrogen bonding modifying Morpholino eteplirsen has partially restored function to and stronger charge-charge interactions. Proteins that have evolved the dystrophin protein, enough to show significant clinical benefit on to bind nucleic acids stabilize binding by attraction between cationic a six-minute walk test [5] versus the untreated control group. amino acids of the protein and the anionic charge on the backbone Introduction to the Molecules phosphates of the nucleic acid, along with weaker hydrogen-bonding interactions. Morpholinos arriving at the active site of a nucleic-acid Morpholino oligos are uncharged analogs of nucleic acids. Typical binding protein will not be held by the charge-charge interactions Morpholino oligos are from 18-30 subunits long, with a nucleic acid because the Morpholino backbone has no charge. This leaves base on each subunit. They are used to alter gene expression by binding hydrogen bonding, which is insufficient force to hold a Morpholino to complementary targets on RNA molecules and blocking processes. in the catalytic site of a nuclease or bind it into a Toll-like receptor. The interactions of Morpholinos with proteins are very weak so they are not cleaved by nucleases and have little effect on cells apart from Morpholinos do not bind to serum, which fits with our hypothesis their RNA binding activity. The structure, design and characteristics that the Morpholino’s lack of electrostatic charge prevents protein of Morpholino oligos have been reviewed elsewhere [6]. binding. Morpholino-serum interaction was assessed by surface plasmon resonance spectroscopy and no significant binding was Morpholino oligos are referred to by a variety of names and detected (Hong Moulton, Pers. Comm). As a result, unmodified acronyms. We usually call them Morpholinos, but in the literature Morpholino oligos have a short serum half-life relative to other oligo they are called Phosphorodiamidate Morpholino Oligos (PMO), types. While the exposure time of cells to Morpholino in the blood Morpholino Oligos (MO), Morpholino Antisense Oligos (MASO) and is relatively short, the oligos are free in solution and available for cell Neu-genes. Variations include delivery-enabled Vivo-Morpholinos, interactions as they are not associated with serum protein. photocleavable Photo-Morpholinos, and cell-penetrating Peptide- conjugated Phosphorodiamidate Morpholino Oligos (PPMO and Stability pip-PMO). The complete resistance of Morpholinos to nucleases has been shown in a study exposing the oligos to a range of nucleases Mechanism of Action and proteases for up to five hours in cell-free enzyme systems [7] Antisense oligos are molecules that bind to RNA by base pairing and by exposure to liver lysates [8]. No degradation of oligos was and change gene expression. The region of RNA complementary to detected in these tests. Later work exposed Morpholino-peptide an antisense oligo is called a target. The sequence of an mRNA is conjugates to serum and living cells. After varying incubation called the sense sequence. times, the Morpholinos were extracted and assessed by MALDI- TOF spectrometry. While intermediate and complete degradation Some antisense oligos rely on the activity of other molecules products of the peptide component were detected, no degradation of such as proteins for their effects