Myotonic dystrophy protein kinase gene expression in skeletal muscle from congenitally affected infants

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

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.     

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.     

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.     

Myotonic dystrophy: relative sensitivity of symptoms signs and abnormal investigations

Twenty-five symptoms, signs, and abnormal investigations were looked for in 20 patients with clinically-definite myotonic dystrophy. Weakness of facial muscles, neck flexors, and arm external rotators was found in all patients (sensitivity = 100%). Arm external rotation has not been reported as a frequently involved muscle in previous clinical studies on myotonic dystrophy. Careful examination of muscle strength may therefore predict which patients may or may not carry the abnormal gene for myotonic dystrophy.     

Myotonic muscular dystrophy: defective phospholipid metabolism in the erythrocyte plasma membrane

Myotonic muscular dystrophy (MyD) is a systemic genetic disorder that is thought to result from a generalized cellular membrane defect although the exact nature of this defect is unknown. This study examines two calcium-dependent membrane processes that have been observed in erythrocytes from healthy individuals: calcium-stimulated phosphatidic acid accumulation and calcium-induced potassium leak. We find that erythrocytes from MyD patients, in contrast to controls, have markedly impaired phosphatidic acid accumulations while maintaining normal potassium leaks. The calcium uptakes and ATP contents of MyD erythrocytes are not different from controls. We conclude that phospholipid metabolism is altered in MyD erythrocytes. The specificity of this abnormality and its relationship to altered muscular function are not known.     

Myotonic dystrophy is a significant cause of idiopathic polyhydramnios

OBJECTIVE: Myotonic dystrophy, the most common form of muscular dystrophy seen in pregnant women, may be a significant cause of middle trimester polyhydramnios. Our purpose was to determine the prevalence of myotonic dystrophy in women with idiopathic polyhydramnios and to characterize the ultrasonographic findings associated with cases. STUDY DESIGN: We examined the cases of 67 patients who were delivered of infants at the University of Utah between 1992 and 1996 with a diagnosis of idiopathic polyhydramnios (amniotic fluid index >25). Women with diabetes mellitus, hydrops, or fetal anomalies known to cause polyhydramnios were excluded from the study. Amniotic fluid samples or cord blood samples were obtained from 41 patients, and polymerase chain reaction amplification and Southern blot analysis were performed to detect the presence of the myotonic dystrophy mutation. Ultrasonographic findings, prenatal course, and neonatal outcomes were reviewed in all cases. RESULTS: Four of the 41 patients tested had the myotonic dystrophy mutation, yielding a prevalence in our population of 9.7%. Three of the 4 patients reported a family history of myotonic dystrophy. Ultrasonographic findings associated with a positive result included abnormal posturing of extremities (3/4) and unilateral clubbed foot (3/4). No other structural or growth abnormalities were seen. Two of the patients were delivered before term, 1 at 26 weeks and 1 at 32 weeks. Three of the 4 infants were severely affected, necessitating admission to the intensive care unit, and 1 died on day 11 after birth. One infant, whose myotonic dystrophy mutation consisted of between 800 and 900 triplet repeats, did not require admission to the intensive care unit. CONCLUSION: Myotonic dystrophy may be seen as idiopathic polyhydramnios and should be considered as part of the differential diagnosis in these cases. Women with a familial history of myotonic dystrophy or ultrasonographic evidence of hypotonia, including positional abnormalities of the extremities, should be offered deoxyribonucleic acid testing for the myotonic dystrophy mutation.     

Comparative gene expression profiling by oligonucleotide fingerprinting

The use of hybridisation of synthetic oligonucleotides to cDNAs under high stringency to characterise gene sequences has been demonstrated by a number of groups. We have used two cDNA libraries of 9 and 12 day mouse embryos (24 133 and 34 783 clones respectively) in a pilot study to characterise expressed genes by hybridisation with 110 hybridisation probes. We have identified 33 369 clusters of cDNA clones, that ranged in representation from 1 to 487 copies (0.7%). 737 were assigned to known rodent genes, and a further 13 845 showed significant homologies. A total of 404 clusters were identified as significantly differentially represented (P < 0.01) between the two cDNA libraries. This study demonstrates the utility of the fingerprinting approach for the generation of comparative gene expression profiles through the analysis of cDNAs derived from different biological materials.     

Fluorescence in situ hybridization analysis of the replication properties of the myotonic dystrophy protein kinase (DMPK) gene region

Myotonic dystrophy (DM) is caused by an expansion of a CTG repeat sequence in the 3' noncoding region of a protein kinase gene (DMPK) at 19q13.3. We used in situ hybridization to analyse the replication timing of the genomic region containing DMPK in fibroblasts and myoblasts from controls and myotonic dystrophy patients. In this method the relative proportion of singlet to doublet hybridization signals is used to infer the relative time of replication of specific loci or regions. Our results show that in cells from normal individuals approximately 65% of signals appear as doublets, indicating early replication. In DM patients with a number of CTG repeats ranging from about 600-1800 we observed a significant increase of singlet-doublets compared to the background level. These results suggest the existence of replication alternations and/or structural differences between the normal and mutant alleles induced by the presence of the DM mutation.     

Recent status of the antisense oligonucleotide approaches in oncology

Antisense oligonucleotides designed to complement a region of a particular messenger RNA may inhibit gene expression potentially through sequence-specific hybridization. Their inhibiting effect has been shown in a variety of in vitro and in vivo models in oncology, whereas much rarer clinical trials have been carried out. Rigorous demonstration of in vitro and in vivo specific effects upon their targets is mandatory before their use as drugs in cancer therapy.     

Pharmacokinetics and metabolism in mice of a phosphorothioate oligonucleotide antisense inhibitor of C-raf-1 kinase expression

The plasma and tissue disposition of CGP 69846A (ISIS 5132) was characterized in male CD-1 mice following iv bolus injections administered every other day for 28 days (total of 15 doses). The doses ranged from 0.8 mg/kg to 100 mg/kg. Urinary excretion of oligonucleotide was also monitored over a 24-hr period following single dose administration over the same dose range. Pharmacokinetic plasma profiles were determined following single dose administration (dose 1) and after multiple doses (dose 15) at doses of 4 and 20 mg/kg. Concentrations in kidney, liver, spleen, heart, lung, and lymph nodes were characterized following doses 1, 8, and 15 for all doses. Capillary gel electrophoresis was used to quantitate intact (full-length) oligonucleotide and its metabolites (down to N - 11 base deletions) in both plasma and tissue at all time points. The plasma and tissue disposition of CGP 69846A was characterized by a rapid distribution into all tissues analyzed. Rapid plasma clearance of the parent oligonucleotide (9.3-14.3 ml/min/kg) was predominantly the result of distribution to tissue and, to a lesser extent, metabolism. Appearance and pattern of chain-shortened metabolites seen in plasma and tissue were consistent with predominantly exonuclease-mediated base deletion. No measurable accumulation of oligonucleotide was observed in plasma following multiple-dose administration, but both the liver and the kidney exhibited 2-3-fold accumulations. In general, the tissues exhibited half-lives for the elimination of parent oligonucleotide of 16-60 hr compared with plasma half-lives of 30-45 min. After repeated administrations, significant decreases in plasma clearance and volume of distribution at steady state (Vss) were observed following dose 15 at the dose of 20 mg/kg but not at the dose of 4 mg/kg. Changes in tissue accumulation and evidence for saturation of tissue distribution at the high doses may explain the plasma disposition changes observed in the absence of alteration of metabolism or plasma accumulation. Urinary excretion was a minor pathway for elimination of oligonucleotide over the 24-hr period immediately following iv administration. However, the amount of oligonucleotide excreted in the urine increased as a function of dose from less than 1% to approximately 13% of the administered dose over a dose range of 0.8 mg/kg to 100 mg/kg.     

Studies on inhibition of activated oncogene expression by antisense RNA]

Blood group ABO antigens are known to be carried by several platelet glycoproteins (GP), e.g. GPIb, GPIIa, GPIIb, GPIIa and PECAM. Beside these proteins, we recently observed that blood group A antigen was also expressed on some other uncharacterized platelet proteins (70-90 kDa) having electrophoretic mobilities closely resembling those of GPIV and GPV. These findings prompted us further to characterize these latter ABO-expressing platelet proteins. By antigen capture ELISA, wherein the monoclonal antibodies (mAbs) CLB-IVC7 and CLB-SWI6 were used to hold the corresponding antigens GPIV and GPV, human anti-A specifically bound to these proteins derived from A1-platelets; neither GPIV nor GPV derived from A2-, B- or O-platelets bound anti-A. In a Western blot assay using immunoprecipitated GPIV and GPV as antigens, mAb anti-A immunostained GPIV and GPV precipitated from A1, but not from A2 and O platelets. These results conclusively demonstrate that blood group A antigen is expressed on platelet GPIV and GPV.     

Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.     

SupraMolecular BioVectors (SMBV) improve antisense inhibition of erbB-2 expression

New therapeutic strategies are now being developed against adenocarcinoma associated with erbB-2 amplification, particularly by inhibiting p185erbB-2 expression. Antisense oligodeoxynucleotides seem promising for this purpose as long as they are efficiently protected against degradation and targeted into the cells. We present antisense oligonucleotide carriers, the supramolecular biovectors (SMBVs), for which we have already demonstrated the ability to improve both cellular uptake and protection of oligodeoxynucleotide. The present work demonstrates that SMBVs elicit a specific and non-toxic action of antisense compounds in a cell model, irrespective of their sensitivity to nucleases. This is a major point, considering the specificity problems associated with the use of nuclease-resistant phosphorothioate oligodeoxynucleotide. SMBVs improve antisense efficiency of oligodeoxynucleotide designed against p185erbB-2, with a complete growth arrest of SK-Br-3, human adenocarcinoma mammary cells that overexpress p185erbB-2 and no effect on MCF-7 cells that normally express p185erbB-2. The comparison of SMBVs with DOTAP reveals the statistically higher efficiency of SMBVs, which allows the antisense inhibition of p185erbB-2 expression in 65-75% of SK-Br-3 cells (P < 0.05). The efficiency and controlled synthesis of SMBVs underline their potentialities as oligodeoxynucleotide carriers for in vivo experiments.