Guide for Morpholino Users: Toward Therapeutics

Special Article - Antisense Drug Research and Development

J Drug Discov Develop and Deliv. 2016; 3(2): 1023.

Guide for Morpholino Users: Toward Therapeutics

Moulton JD*

Gene Tools, LLC, USA

*Corresponding author: Moulton JD, Gene Tools, LLC, 1001 Summerton Way, Philomath, Oregon 97370, USA

Received: January 28, 2016; Accepted: April 29, 2016; Published: May 03, 2016


Morpholino oligos are uncharged molecules for blocking sites on RNA. They are specific, soluble, non-toxic, stable, and effective antisense reagents suitable 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 translation, modifying splicing of pre-mRNA, inhibiting miRNA maturation and 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, Therapeutic Promise

Morpholino oligos bind to complementary sequences of RNA and get in the way of processes. Morpholino oligos are commonly used to prevent a particular protein from being made in an organism 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 mutant (called a gene knockout) or by interrupting processes on RNA (called a gene knockdown). Some DNA alterations cause production of a protein to decrease without stopping all production; confusingly, these are also called gene knockdowns. DNA alterations are permanent, while knockdowns of RNA are either transient, generally last several days after dosing with antisense (such as Morpholinos), or are long-term, depending on continued production of knockdown RNA in cells (such as shRNA transcribed in cells from a plasmid).

Once introduced into cells, Morpholinos freely diffuse between the cytosol and nuclear compartments and bind complementary sequences of RNA. Morpholinos have been used as gene knockdown reagents in cell cultures and in animals, bacteria, protists, plants and fungi. The RNA-blocking property of Morpholinos has also been used to cause a range of different outcomes beyond simple gene knockdowns. Morpholinos have different effects depending on the sort of target they bind. Morpholinos can:

Morpholinos have been broadly used in the developmental biology community to knock down genes in embryos of organisms such as zebrafish (Danio rerio), African clawed frogs (Xenopus sp.), chicks (Gallus gallus), sea urchins (e.g. Strongylocentrotus sp.), sea squirts (Ciona sp.), and many more. The oligos are usually microinjected though fine glass needles into early embryos at the one-to-few cell stage. Many kinds of antisense have toxic effects during development of an embryo. Because Morpholinos have little interaction with protein they are unusually non-toxic antisense, sufficiently non-toxic to make them the first choice of most developmental biologists for transient gene knockdowns. In contrast, injection of oligos containing phosophorothioate intersubunit linkages often kills embryos [1]. Morpholinos are highly specific antisense, having less interaction with unintended RNAs than antisense which employs protein activity; this is because a Morpholino must be complementary to a longer sequence of RNA than antisense using catalytic activity (e.g. RNAi, phosphorothioate RNA, etc.) [2]. Less specific antisense causes changes in gene expression during development of the embryo and can cause developmental defects (teratogenesis) [3].

If you want to use an antisense oligo for a therapeutic, nontoxicity and specificity are excellent characteristics with which to start. Morpholinos are also reasonably water soluble, with most oligos staying in solution for many months to years at 1mM concentration in room temperature water; this gives them a great advantage over another kind of potential therapeutic antisense, the Peptide Nucleic Acids (PNA) [4]. Clinical trials conducted by Sarepta Theraeputics Inc. (formerly AVI BioPharma Inc.) have shown excellent safety of the oligos in humans (Table 1).

Citation: Moulton JD. Guide for Morpholino Users: Toward Therapeutics. J Drug Discov Develop and Deliv. 2016; 3(2): 1023. ISSN : 2471-0288