Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc.

The phosphorodiamidate Morpholino oligomers (PMO) are a new class of antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. In a search for a Morpholino agent that would inhibit cell proliferation, it was found that oligomers directed against c-myc, a gene involved in control of the cell cycle, were effective. The sequence specificity and mechanism of action of one agent were determined. The 20-mer 126 lowers c-myc protein levels in treated cells and arrests cells in G0/G1 of the cell cycle. It also acts at the RNA level to inhibit normal pre-mRNA splicing and instead produces an aberrantly spliced mRNA. Irrelevant and mispair control oligomers indicated that the observed antiproliferative effect was sequence specific. This was confirmed in a reporter gene model system using a c-myc 5'-untranslated region (5'-UTR) fused to a cDNA copy of the insect luciferase gene. We conclude that 126 is acting through an antisense mechanism involving Watson-Crick hydrogen bonding to its target RNA. A specific antisense agent directed against a cell cycle-associated gene mRNA may be useful as a therapeutic in diseases characterized by excess cell proliferation, such as restenosis following balloon angioplasty or cancer.

c-Myc antisense limits rat liver regeneration and indicates role for c-Myc in regulating cytochrome P-450 3A activity.

Expression of c-myc protein is associated with cell proliferation. The present study uses antisense oligomers to inhibit c-myc expression in the regenerating rat liver after 70% partial hepatectomy (PH). Antisense phosphorodiamidate morpholino oligomers (novel DNA analogs) were administered i.p. immediately after surgery to block expression of c-myc within the first 24 h after PH. A 20-mer PMO complimentary to the c-myc mRNA at the translation start site was an effective sequence (AVI-4126, 5'-ACGTTGAGGGGCATCGTCGC-3'). A single i.p. dose of 0.5 mg/kg AVI-4126 caused reduction of the regenerating liver c-myc protein in a sequence-specific and dose-dependent manner. Inhibition of c-myc expression resulted in reduction of proliferating cell nuclear antigen and arrested cells in the G(0)/G(1) phase of the cell cycle. The ratio of G(2):G(0) cell populations in the regenerating liver 24 h after PH dropped from 29.1 in saline vehicle-treated rats to 18.0 in rats treated with 2.5 mg/kg AVI-4126. The expression of cell cycle checkpoint protein p53 was inhibited with increasing doses of AVI-4126, but expression of p21(waf-1) was unaffected. The activity of cytochrome P-450 3A2 (CYP3A2) was evaluated by immunoblot analysis and erythromycin N-demethylation. AVI-4126 did not alter CYP3A activity in nonhepatectamized animals but showed a dose-dependent decrease in PH rats. We conclude that AVI-4126, antisense oligomer to c-myc, can reduce cell proliferation in the regenerating rat liver. Furthermore, inhibition of c-myc may indirectly influence the expression of CYP3A.

Effect of TNF-alpha antisense oligomers on cytokine production by primary murine alveolar macrophages.

Antisense oligomers can inhibit expression of a single gene in a sequence-specific manner. As a result, these sequences are being developed both as powerful experimental tools in the laboratory and as a novel class of therapeutic agents. In this study, we evaluated a panel of morpholino antisense (M-AS) oligomers for their ability to inhibit tumor necrosis factor-alpha (TNF-alpha) production by primary murine alveolar macrophages (AMs) and compared them with the more commonly used phosphorothioate oligonucleotides (S-AS). We found that 25 microM of morpholino oligomers whose sequence spanned the AUG (M-AS 2, M-AS 2me, and M-AS 5) start codon of TNF-alpha significantly inhibited TNF production on stimulation by both lipopolysaccharides (LPS) (36.6 +/- 3.2%, 27.3 +/- 3.0%, and 37.7 +/- 2.0% inhibition, respectively), whereas S-AS targeted toward the same region were ineffective. M-AS 2 and M-AS 2me also significantly inhibited TNF production in AMs stimulated by adherence to a solid substrate (28.7 +/- 2.2% and 29.4 +/- 8.3% inhibition, respectively). Increasing the concentration of M-AS 2 and M-AS 2me to 50 microM improved their efficacy in both LPS-stimulated (42.7 +/- 1.5% and 45.9 +/- 2.1% inhibition, respectively) and adherence-stimulated (52.6 +/- 0.7% and 41.7 +/- 2.9% inhibition, respectively) AMs. In contrast, we showed that neither an antisense sequence targeted to a region upstream of the AUG site (M-AS 4) nor the nonsense control sequences M-NS 1 and M-NS 2 significantly inhibited TNF-alpha production by AMs on exposure to either stimulus. The data indicate that morpholino oligomers inhibit TNF-alpha production by murine AMs in a sequence-dependent and dose-dependent manner.

Comparison of efficacy of antisense oligomers directed toward TNF-alpha in helper T and macrophage cell lines.

The authors investigated the use of antisense oligomers specific for TNF-alpha (AS-2) and nonsense control oligomers (NS) in T cells (HT2) and macrophages (RAW264.7), comparing three distinct chemical formulations. Phosphorothioate antisense (S-AS) caused sequence-specific inhibition of TNF-alpha production by activated HT2s (0.5 microM S-AS 2 vs S-NS: 31.4 +/- 1.2%, 4.2 +/- 3.2% inhibition, respectively). In contrast, S-AS were ineffective in RAW264.7, despite greater uptake as measured with fluorescent S-oligonucleotides. Furthermore, differences in efficacy of S-AS (HT2 > RAW) were not attributable to differences in the pinocytic (HT2 = RAW) or adsorptive endocytic (RAW > HT2) pathways implicated in oligonucleotide uptake, suggesting an important role for intracellular events after antisense uptake. Morpholino oligomers (M-AS), in contrast, were more effective in RAW264.7 than in HT2 (32.6 +/- 2.6% vs 12.3 +/- 0.5% inhibition), consistent with uptake experiments using fluorescent M-oligomers. Phosphodiester oligonucleotides were ineffective in both cell types. It was concluded that antisense efficacy in leukocytes varies according to type of oligomer, cell target and intracellular processing event(s).

In vitro efficacy of morpholino-modified antisense oligomers directed against tumor necrosis factor-alpha mRNA.

Chemical modification of antisense oligonucleotides to increase nuclease resistance may improve their efficacy within enzyme-rich cellular targets (e.g. macrophages). We evaluated a panel of morpholino antisense oligomers (M-AS) for their ability to inhibit macrophage tumor necrosis factor-alpha (TNF-alpha) release and compared them to phosphodiester (O-AS) and phosphorothioate (S-AS) types of oligonucleotides. M-AS inhibited translation in vitro (rabbit reticulocyte lysate) of target mRNA at concentrations as low as 200 nM (e.g. percent inhibition by M-AS 2 at 0.2, 1.0, and 2.0 microM was 40.9 +/- 5.3%, 50.2 +/- 4.6%, and 57.7 +/- 3.6%, respectively, n = 4, p

Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation.

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.

Investigations of oligodeoxyinosine for triple helix formation.

The ability of a 17-mer of deoxyinosine to form a triple helix with a (dT)17(dA)17 segment of a 29-mer Watson-Crick duplex was investigated. Under conditions amenable to triple helix formation, (dT)17 showed evidence of complex formation as evidenced by UV melting curves and gel mobility shift assays whereas (dI)17 did not.

Uncharged stereoregular nucleic acid analogs: 2. Morpholino nucleoside oligomers with carbamate internucleoside linkages.

A novel oligonucleotide analog has been prepared from ribonucleoside derived morpholine subunits linked by carbamate groups. Oxidative cleavage of the 2',3' vicinal diol of cytidine followed by reductive amination of the resulting dialdehyde afforded the morpholine subunit. Coupling of the subunits are through carbamate moieties and the oligomers were characterized by 1H NMR and FAB MS. Evidence for interaction of the hexamer 19 with p(dG6) was found, but an atypical interaction of 19 with a RNA target was observed.

Carbon-13 nuclear magnetic resonance assignments of pactamycin and related compounds.

All resonances observed in the 13C NMR spectrum of the antitumor antibiotic pactamycin and its degradation product pactamyçate have been assigned, employing off-resonance and specific proton decoupling as well as comparison with the 13C NMR spectra of the model compounds m-aminoacetophenone and ethyl 6-methylsalicylate.

A practical synthesis of codeine from dihydrothebainone.

The conversion of dihydrothebainone to codeine or thebaine has been achieved in high yield. Bromination and dehydrobromination constructs the 4,5-oxide bridge to give 1-bromo- and 1,7-dibromodihydrocodeinone which yield dihydrocodeinone practically quantitatively after catalytic debromination. Ketalization and acid-catalyzed elimination of methanol give excellent yields of delta6-dihydrothebaine to which is added methyl hypobromite using N-bromoacetamide in methanol. The action of potassium tert-butoxide in Me2SO on the resulting 7-bromodihydrocodeinone dimethyl ketal gives codeinone dimethyl ketal selectivity at 60 degrees while at 120 degrees thebaine is the exclusive product. Hydrolysis to codeinone and borohydride reduction give codeine in 70% overall yield. The bromo intermediates in the formation of the 4,5-oxide bridge have been examined. 1,5beta, 7alpha-Tribromodihydrothebainone has been identified as the main product in the tribromination of dihydrothebainone.