Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

Elucidation of gene function using C-5 propyne antisense oligonucleotides

Identification of human disease-causing genes continues to be an intense area of research. While cloning of genes may lead to diagnostic tests, development of a cure requires an understanding of the gene's function in both normal and diseased cells. Thus, there exists a need for a reproducible and simple method to elucidate gene function. We evaluate C-5 propyne pyrimidine modified phosphorothioate antisense oligonucleotides (ONs) targeted against two human cell cycle proteins that are aberrantly expressed in breast cancer: p34cdc2 kinase and cyclin B1. Dose-dependent, sequence-specific, and gene-specific inhibition of both proteins was achieved at nanomolar concentrations of ONs in normal and breast cancer cells. Precise binding of the antisense ONs to their target RNA was absolutely required for antisense activity. Four or six base-mismatched ONs eliminated antisense activity confirming the sequence specificity of the antisense ONs. Antisense inhibition of p34cdc2 kinase resulted in a significant accumulation of cells in the Gap2/mitosis phase of the cell cycle in normal cells, but caused little effect on cell cycle progression in breast cancer cells. These data demonstrate the potency, specificity, and utility of C-5 propyne modified antisense ONs as biological tools and illustrate the redundancy of cell cycle protein function that can occur in cancer cells.     

A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides

An enzyme competitive hybridization assay was developed and validated for determination of mouse plasma concentrations of a 15mer antisense phosphodiester oligodeoxyribonucleotide and of two phosphorothioate analogs. Assays were performed in 96-well microtiter plates. The phosphodiester sense sequence was covalently bound to the microwells. The 5'-biotinylated antisense sequence was used as tracer. The principle of the assay involves competitive hybridization of tracer and antisense nucleotide to the solid phase-immobilized sense oligonucleotide. Solid phase- bound tracer oligonucleotide was assayed after reaction with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. As in competitive enzyme immunoassays, coloration was inversely related to the amount of analyte initially present in the sample. The limit of quantification was 900 pM for phosphodiester antisense oligonucleotide using a 100 microl volume of plasma without extraction. Cross-reactivity was negligible after a four base deletion in either the 3'or 5'position. The assay was simple and sensitive, suitable for in vitro screening of oligonucleotide hybridization potency in biological fluids and for measuring the plasma pharmacokinetics of phosphorothioate and phosphodiester sequences.     

Transient c-fos gene expression in cerebellar development and functional stimulation

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/ reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

An in vitro messenger RNA binding assay as a tool for identifying hybridization-competent antisense oligonucleotides

Targeting, use of appliances, and standards of outcome for General Dental Service orthodontic cases collected between 1990 and 1991 were compared with a sample of cases from an earlier study, collected between 1987 and 1988, using the PAR index and IOTN. Comparisons are made generally and in relation to the changes in prior approval regulations for cases started since October 1987. More lower-need cases were treated, but there were no more unnecessary' treatments and there has been a limited improvement in outcomes, as assessed by the indices, associated with increased use of fixed appliances since 1987. Prior approval appeared to give no tangible benefits in terms of levels of unnecessary treatment or quality of outcome.     

Low-cost system for real-time monitoring of luciferase gene expression

In some mammalian cells transfected with luciferase reporter genes, the luciferase/luciferin reaction in a cell monolayer produces a very small light flux. While the low light levels are often measurable with single-photon counting cameras, these devices are expensive and may require long averaging times to acquire an image. We describe an approach for real-time monitoring of light produced by luciferase gene expression in intact, cultured cells using readily available and relatively inexpensive components. The system uses a single-photon counting photomultiplier tube with built-in high voltage supply and photon counting circuitry to rapidly measure average light output from growing cells in a 35 mm culture dish. The fast, accurate and highly sensitive response of the system makes it useful for studying the dynamics of gene expression over time periods ranging from minutes to days.     

Myotonic dystrophy: antisense oligonucleotide inhibition of DMPK gene expression in vitro

Antisense phosphorothioate oligonucleotides, targeted against the first codon starting region of DMPK mRNA, were successfully used in K562 and HepG2 cells to decrease DMPK expression. The most effective antisense oligo, MIO1, when added to K562 cells, shows a 75% reduction of the DMPK gene expression 6 hours after addition. The same molecule, when encapsulated in liposomes, delays myotonin mRNA decrease at 24 hours after cell treatment. This considerable success with such inhibition in vitro could be utilised to generate a cell model to study myotonic dystrophy (DM) chemio-physiological alterations.     

Sustained reduction of neointima with c-myc antisense oligonucleotides in saphenous vein grafts

BACKGROUND: Treatment of saphenous veins with c-myc antisense oligomers during preparation for grafting reduces medial cellular proliferation and macrophage infiltration, and preserves medial smooth muscle content at 3 days. Accordingly, the purpose of this study was to examine whether c-myc antisense oligomers have an impact on late vein graft remodeling. METHODS: Sixty-two pigs underwent unilateral saphenous vein-carotid artery interposition grafting. Harvested veins were incubated either in saline (control group) or 20-micromol/L or 200-micromol/L concentrations of c-myc antisense oligomers (treated groups) for 30 minutes intraoperatively. Three months after surgery, vein graft histology was assessed. RESULTS: Forty-five of 62 randomized animals survived the experiment; no differences in animal survival or graft patency among the groups were observed (p = NS, chi2). C-myc antisense oligomers significantly decreased neointimal and wall thickness, as well as increased lumenal index, in treated groups (p<0.04, p<0.03, and p<0.001, respectively, analysis of variance). In contrast, there was no difference in medial thickness or perivascular wound healing. CONCLUSION: Intraoperative treatment of saphenous veins with c-myc antisense oligomers decreased neointimal formation at 3 months after grafting. In conjunction with our previous reports, these findings suggest that early inhibition of cellular proliferation and inflammatory infiltration results in a sustained reduction in neointimal formation and favorable graft remodeling.     

Modeling stochastic gene expression: implications for haploinsufficiency

There is increasing recognition that stochastic processes regulate highly predictable patterns of gene expression in developing organisms, but the implications of stochastic gene expression for understanding haploinsufficiency remain largely unexplored. We have used simulations of stochastic gene expression to illustrate that gene copy number and expression deactivation rates are important variables in achieving predictable outcomes. In gene expression systems with non-zero expression deactivation rates, diploid systems had a higher probability of uninterrupted gene expression than haploid systems and were more successful at maintaining gene product above a very low threshold. Systems with relatively rapid expression deactivation rates (unstable gene expression) had more predictable responses to a gradient of inducer than systems with slow or zero expression deactivation rates (stable gene expression), and diploid systems were more predictable than haploid, with or without dosage compensation. We suggest that null mutations of a single allele in a diploid organism could decrease the probability of gene expression and present the hypothesis that some haploinsufficiency syndromes might result from an increased susceptibility to stochastic delays of gene initiation or interruptions of gene expression.     

Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay

Blood samples and questionnaire background data were collected from 96 children (age 2-14 years) living in urban, suburban, or rural areas with varying traffic intensity and industrial lead pollution in Uruguay. Spot samples of tap water were collected from the homes of 44 children, and samples of top soil were taken from seven areas. Samples of air-borne dust were collected in central and suburban Montevideo. Blood lead concentrations (B-Pb) in children ranged between 47 and 191 (mean 96) micrograms/L and exceeded in 36% of the children 100 micrograms/L, the intervention level adopted by the United States Centers for Disease Control. Lead in tap water ranged from 0.2 to 230 (mean 15) micrograms/L and exceeded in 39% of the samples the maximum level recommended by WHO, 10 micrograms/L. Lead pipes were used in parts of the water supply systems. Lead in air varied between different locations from 0.15 to 1.7 micrograms/m3, highest in the very center of Montevideo. The median soil lead ranged from 6 to 2100 micrograms/g and was highest in industrially polluted areas. At multiple regression analysis, B-Pb was significantly associated only with age (P = 0.032) and traffic intensity at school (P = 0.045). No significant impact on B-Pb of lead in water or soil could be established.     

Inhibition of N-methyl-D-aspartate glutamate receptor subunit expression by antisense oligonucleotides reveals their role in striatal motor regulation

N-methyl-D-aspartate (NMDA) glutamate receptors have an established role in the regulation of motor behavior by the basal ganglia. Recent studies have revealed that NMDA receptors are heteromeric assemblies of structurally related subunits from two families: NMDAR1, which is required for channel activity, and NMDAR2A-D, which modulate the properties of the channels. In the rat, the NMDA receptor subunits exhibit anatomically restricted patterns of expression, so that each component of the basal ganglia has a distinct NMDA receptor subunit mRNA phenotype. We have used in vivo intrastriatal injection of synthetic antisense oligodeoxynucleotides (ODNs) to examine the roles of particular NMDA receptor subunits in the regulation of motor behavior in rats. Injection of 15 nmol of a 20-mer ODN targeted to the NMDAR1 subunit induced spontaneous ipsilateral rotation. Smaller doses of NMDAR1 antisense ODN did not lead to spontaneous rotation, but prominent ipsilateral rotation was observed after systemic administration of D-amphetamine. An antisense ODN to NMDAR2A was also effective in eliciting amphetamine-inducible rotation, although the magnitude of the effect was less than that seen with NMDAR1, whereas ODNs targeted to NMDAR2B, NMDAR2C and an NMDAR1 sense strand ODN had no effect on behavior. In situ hybridization demonstrated that injection of the NMDAR1, NMDAR2A or NMDAR2B antisense ODNs produced specific reductions in target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate to NMDA sites was not altered, although strychnine-insensitive 3H-glycine binding sites exhibited a small but significant reduction. These observations suggest that NMDA receptor complexes containing NMDAR1 and, to a lesser extent, NMDAR2A subunits play particularly important roles in the regulation of motor behavior by neostriatal neurons.     

Potent and selective inhibition of gene expression by an antisense heptanucleotide

Factors that govern the specificity of an antisense oligonucleotide (ON) for its target RNA include accessibility of the targeted RNA to ON binding, stability of ON/RNA complexes in cells, and susceptibility of the ON/RNA complex to RNase H cleavage. ON specificity is generally proposed to be dependent on its length. To date, virtually all previous antisense experiments have used 12-25 nt-long ONs. We explored the antisense activity and specificity of short (7 and 8 nt) ONs modified with C-5 propyne pyrimidines and phosphorothioate internucleotide linkages. Gene-selective, mismatch sensitive, and RNase H-dependent inhibition was observed for a heptanucleotide ON. We demonstrated that the flanking sequences of the target RNA are a major determinant of specificity. The use of shorter ONs as antisense agents has the distinct advantage of simplified synthesis. These results may lead to a general, cost-effective solution to the development of antisense ONs as therapeutic agents.     

Induction of apoptosis by organotin compounds in vitro: neuronal protection with antisense oligonucleotides directed against stannin

Immortalized cell lines and primary neuronal cultures were used to characterize the selective toxicity of trimethyltin (TMT),triethyltin (TET) and tributyltin (TBT). TBT and TET were cytotoxic at similar concentrations in the immortalized cell lines tested; the 50% toxic concentration (TC50) was 1 to 11 microM. In contrast, immortalized cell lines varied considerably in their sensitivity to TMT, with sensitive cell lines (neuroblastomas, T-, B-cell lines) showing TC50 values of 2 to 8 microM, whereas insensitive cells (NIH-3T3 fibroblast, HTB-14 glioma, TC-7 kidney cells) had TC 50 values > 100 microM. Primary neuronal cell cultures were very sensitive to organotins (TC50 values, 1-10nM), and showed patterns of selective toxicity with respect to neuronal and glial cells. Because organotin toxicity evolves over 24 to 48 hr. we determined whether these compounds induced apoptosis in primary cultures. TMT increased (P < .05) the fraction of apoptotic cells 6 and 12 hr after treatment with TMT at TC50 concentrations. Prior studies suggested that a protein, stannin, was localized in cells sensitive to organotins. Stannin was expressed in several TMT-sensitive cell lines (PC12, T, B cells) and in primary neurons in culture. Stannin was absent in the resistant HTB-14 glioma cell line. The role of stannin in mediating TMT toxicity in primary cultures was investigated by blocking stannin expression with specific antisense oligonucleotides. Treatment of primary cultures with antisense oligonucleotides for 48 hr before and during TMT treatment significantly protected neurons from the neurotoxic and apoptotic effects of TMT. This effect was not observed with scrambled oligonucleotide controls. Thus, TMT may induce apoptosis in sensitive cells, which is partly mediated by stannin. Based on the available data we conclude that stannin expression is necessary, but not sufficient for TMT toxicity.     

Growth inhibition of human tumor cell lines by antisense oligonucleotides designed to inhibit p120 expression

Using a rat model of short- (4 weeks) and long-term (10 weeks) ascending aortic banding and debanding, we examined the relationship between coronary dilator reserve and morphological vascular changes. After 4 or 10-week banding, in vivo systolic left ventricular pressure and ventricular wt/body wt ratio increased to a similar level, compared with controls. The coronary dilator reserve measured in an isolated heart preparation decreased similarly in the two banded groups, compared with controls. The ratios of medial to luminal area and perivascular collagen to luminal area in coronary microvessels increased in the banded groups. At 4 weeks after debanding, cardiac hypertrophy regressed to the control level, and the duration of banding did not alter the extent of the regression. The coronary dilator reserve normalized in the group debanded after 4-week banding, but did not regress in the group debanded after 10-week banding. In both of the debanded groups, the hypertrophied media regressed completely. The increased perivascular collagen regressed almost completely in the group debanded after 4-week banding, but remained greater in the group debanded after 10-week banding than in the controls. From these results, we conclude that (i) the regression of medial hypertrophy does not always improve the decreased coronary dilator reserve, and (ii) the vascular fibrosis may be the major cause of the irreversibility of decreased coronary dilator reserve in long-term cardiac hypertrophy.     

Protein kinase A-directed antisense restrains cancer growth: sequence-specific inhibition of gene expression

Increased expression of the RI alpha subunit of cAMP-dependent protein kinase type I has been shown in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. The sequence-specific inhibition of RI alpha gene expression by an antisense oligodeoxynucleotide results in the differentiation of leukemia cells and growth arrest of cancer cells of epithelial origin. A single-injection RI alpha antisense treatment in vivo also causes a reduction in RI alpha expression and inhibition of tumor growth. Tumor cells behave like untransformed cells by making less protein kinase type I. The RI alpha antisense, which produces a biochemical imprint for growth control, requires infrequent dosing to restrain neoplastic growth in vivo.     

Muscle development: electrical control of gene expression

The electrical activity resulting from stimulation by motor neurons regulates gene expression in skeletal muscle fibres. A recent study has suggested a mechanism by which distinct patterns of electrical stimulus might be integrated to control the contractile properties of these fibres.