Previous reports from this laboratory have shown marked cytocidal effects of the ISIS-3466 antisense phosphorothioate oligodeoxynucleotide to the human nucleolar protein p120 on human cancer cell lines in vitro and inhibition of tumor growth in vivo in an i.p/i.p. LOX cell model (L. Perlaky et al. Anti-Cancer Drug Design 8:3-14, 1993). In this study, light and fluorescence microscopy showed that the number of LOX cells in mitosis decreased by 50% after incubation for 4 h in 0.2-0.4 microM antisense oligonucleotide; a 70% reduction in cell number was found from 8-72 h post-treatment. In addition, marked unravelling of nucleolar structures and chromatin fragmentation was found after a 4-h incubation. The nucleolar unravelling occurred in varying degrees ranging from partial unfolding to almost complete separation of the strands of nucleolar residues. Twenty four hours post-treatment, immunofluorescence staining with the anti-p120 monoclonal antibody showed reduced nucleolar protein p120 and translocation of the p120 protein from the nucleoli to the nucleoplasm. Analysis of the mechanisms of the nucleolar unravelling and inhibition of mitosis will provide further understanding of the cytocidal effects of the ISIS-3466 antisense oligonucleotide.
- MeSH
- Melanoma, Amelanotic * genetics chemistry pathology MeSH
- Antigens, Neoplasm * drug effects MeSH
- Oligodeoxyribonucleotides, Antisense * MeSH
- Oligonucleotides, Antisense pharmacology genetics MeSH
- Cell Nucleolus * drug effects MeSH
- Cell Nucleus drug effects MeSH
- Chromatin drug effects MeSH
- Microscopy, Fluorescence MeSH
- Nuclear Proteins * drug effects MeSH
- Humans MeSH
- Mitosis drug effects MeSH
- Molecular Sequence Data MeSH
- Tumor Cells, Cultured drug effects MeSH
- Base Sequence MeSH
- Thionucleotides pharmacology genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.
- MeSH
- Oligonucleotides, Antisense * administration & dosage MeSH
- Cell Death MeSH
- Gene Knockdown Techniques methods veterinary MeSH
- Gonads chemistry MeSH
- DNA, Complementary chemistry MeSH
- Morpholinos * administration & dosage MeSH
- RNA-Binding Proteins analysis genetics MeSH
- Fishes * genetics MeSH
- Base Sequence MeSH
- Sequence Alignment MeSH
- Sterilization, Reproductive methods veterinary MeSH
- Germ Cells physiology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Effective translation of rare disease diagnosis knowledge into therapeutic applications is achievable within a reasonable timeframe; where mutations are amenable to current antisense oligonucleotide technology. In our study, we identified five distinct types of abnormal splice-causing mutations in patients with rare genetic disorders and developed a tailored antisense oligonucleotide for each mutation type using phosphorodiamidate morpholino oligomers with or without octa-guanidine dendrimers and 2'-O-methoxyethyl phosphorothioate. We observed variations in treatment effects and efficiencies, influenced by both the chosen chemistry and the specific nature of the aberrant splicing patterns targeted for correction. Our study demonstrated the successful correction of all five different types of aberrant splicing. Our findings reveal that effective correction of aberrant splicing can depend on altering the chemical composition of oligonucleotides and suggest a fast, efficient, and feasible approach for developing personalized therapeutic interventions for genetic disorders within short time frames.
- MeSH
- Oligonucleotides, Antisense * therapeutic use genetics MeSH
- Genetic Diseases, Inborn genetics therapy MeSH
- Humans MeSH
- Morpholinos therapeutic use genetics MeSH
- Mutation * MeSH
- RNA Splicing * MeSH
- Rare Diseases * genetics drug therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Eluforsen is an antisense oligonucleotide designed to bind to the mRNA region around the F508-encoding deletion and restore the cystic fibrosis transmembrane conductance regulator (CFTR) protein function in the airway epithelium. We assessed the safety and tolerability, pharmacokinetics and exploratory measures of efficacy of inhaled eluforsen in cystic fibrosis (CF) patients homozygous for the F508del-CFTR mutation. METHODS: This randomised, double-blind, placebo-controlled, dose escalation 1b study recruited adult CF subjects with a FEV1 > 70% predicted in four single ascending dose cohorts and four multiple ascending dose cohorts. Primary objectives were safety and tolerability. Secondary endpoints included pharmacokinetics, percent predicted forced expiratory volume in 1 s (ppFEV1), and Cystic Fibrosis Questionnaire-Revised (CFQ-R) Respiratory Symptom Score (RSS). RESULTS: Single and multiple doses of inhaled eluforsen up to 50 mg were safe and well tolerated. A maximum tolerated dose was not established. Systemic exposure was low in all cohorts and lung function remained stable throughout the study. Three of four eluforsen-treated groups in the MAD study demonstrated an improvement in CFQ-R RSS at end of treatment with adjusted mean change from baseline values ranging from 6.4 to 12.7 points. In comparison, there was a mean decrease of 6.5 points in the placebo group from baseline to end of treatment. CONCLUSIONS: Inhaled eluforsen up to 50 mg dosed 3 times per week for 4 weeks was safe and well tolerated, showed low systemic exposure, and demonstrated improvement in CFQ-R RSS, a relevant measure of clinical benefit in CF patients.
- MeSH
- Oligonucleotides, Antisense administration & dosage adverse effects MeSH
- Administration, Inhalation MeSH
- Cystic Fibrosis * drug therapy genetics physiopathology MeSH
- Adult MeSH
- Double-Blind Method MeSH
- Cross-Over Studies MeSH
- Humans MeSH
- Drug Monitoring methods MeSH
- Mutation MeSH
- Oligonucleotides * administration & dosage adverse effects MeSH
- Cystic Fibrosis Transmembrane Conductance Regulator genetics MeSH
- Respiratory Function Tests methods MeSH
- Symptom Assessment methods MeSH
- Treatment Outcome MeSH
- Dose-Response Relationship, Drug * MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Randomized Controlled Trial MeSH
- Research Support, N.I.H., Extramural MeSH
Cationic 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin was tested as a delivery agent for oligonucleotides. By using fluorescence microimaging, it has been shown that complexation of the porphyrin to the phosphorothioate analog of dT(15) labeled by rhodamine enabled its nonendocytic penetration into the cell and regular distribution in the cytoplasm and preferentially into the nucleus. Time-resolved microfluorescence spectroscopy revealed that the oligonucleotide integrity was kept. A small fraction of the porphyrin molecules seems to undergo change of the binding mode after internalization, probably due to duplex formation between the oligonucleotide and its cellular target sequences, or due to dissociation of the porphyrin from the oligonucleotide and subsequent interactions in the cellular environment. (c) 2006 Wiley Periodicals, Inc.
sv.
- MeSH
- Antisense Elements (Genetics) MeSH
- Oligonucleotides, Antisense MeSH
- Gene Expression drug effects MeSH
- Nucleic Acids, Nucleotides, and Nucleosides MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- genetika, lékařská genetika
- biochemie
- biologie
- NML Publication type
- elektronické časopisy
