Dwight D. Weller

Antisense promised major advances in treating a broad range of intractable diseases, but in recent years progress has been stymied by technical problems, most notably inadequate specificity, ineffective delivery into the proper subcellular compartment, and unpredictable activity within cells. Herein is an overview of the design, preparation, and properties of Morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells. Morpholino oligos also exhibit little or no nonantisense activity, afford good water solubility, are immune to nucleases, and are designed to have low production costs.

Oligomers possessing the Morpholino phosphorodiamidate backbone were evaluated for resistance to a variety of enzymes and biologic fluids. A 25-mer was incubated with nucleases, proteases, esterases, and serum, and the reaction mixtures were directly analyzed by MALDI-TOF mass spectrometry. The 25-mer was completely resistant to 13 different hydrolases and serum and plasma. The excellent resistance of Morpholino phosphorodiamidates to enzymatic attack indicates their suitability for in vivo use.

Morpholino and phosphorothioate (S-DNA) antisense oligos were compared in both cell-free and in-cell translation systems. In the most stringent test of specificity in the cell-free system, a globin-targeted S-DNA oligo was found to inhibit its target sequence at concentrations of 10 nM and above, but the sequence-specific component of this inhibition dropped below 50% at concentrations of 100 nM and above. A corresponding Morpholino oligo achieved even higher inhibition at 10 nM, but in contrast to the S-DNA, with the Morpholino, the sequence-specific component of this inhibition remained above 93% at a concentration of 3000 nM. In this same cell-free test system, several S-DNA oligos exhibited substantial undesired nonantisense effects at concentrations of 300 nM and above, whereas corresponding Morpholino oligos exhibited little or no nonantisense activity through a concentration of 3000 nM. In scrape-loaded HeLa cells, both globin-targeted and HBV-targeted S-DNAs (both antisense and control oligos) generally failed to achieve significant translational inhibition at extracellular concentrations up to 3000 nM. In contrast, the Morpholino oligos achieved effective and specific translational inhibition at extracellular concentrations ranging from 30 nM to 3000 nM.

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.

A number of novel phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared by a rapid 2-step procedure, not necessitating prior sugar protection. Spectroscopic and lipophilicity data have been collected on these compounds, and they have been assayed with a range of hydrolytic enzymes. The compounds have been found to be highly resistant to hydrolysis at physiological pH, enzymatic or otherwise. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of these compounds, in the range 3–300μM. Moreover, the degree of inhibition showed a close correlation to chemical structure; in particular, there was a direct relationship between inhibition of thymidine incorporation and lipophilicity. These results suggest cellular penetration by the phosphate triesters and intracellular hydrolysis, by an unspecified mechanism, to the free nucleotide or nucleoside.