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.

• Klíčová slova
• Antisense oligonucleotide, CFQ-R RSS, Clinical trial, Delta F508, Pulmonary medicine,
• MeSH
• antisense oligonukleotidy aplikace a dávkování   škodlivé účinky   MeSH
• aplikace inhalační MeSH
• cystická fibróza * farmakoterapie   genetika   patofyziologie   MeSH
• dospělí MeSH
• dvojitá slepá metoda MeSH
• klinické křížové studie MeSH
• lidé MeSH
• monitorování léčiv metody   MeSH
• mutace MeSH
• oligonukleotidy * aplikace a dávkování   škodlivé účinky   MeSH
• protein CFTR genetika   MeSH
• respirační funkční testy metody   MeSH
• určení symptomu metody   MeSH
• výsledek terapie MeSH
• vztah mezi dávkou a účinkem léčiva * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antisense oligonukleotidy MeSH
• CFTR protein, human MeSH Prohlížeč
• eluforsen MeSH Prohlížeč
• oligonukleotidy * MeSH
• protein CFTR 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.

• Klíčová slova
• Antisense morpholino oligonucleotide, Dead end gene, Germ line chimera, Primordial germ cell, Sterilization, Sturgeon,
• MeSH
• antisense oligonukleotidy * aplikace a dávkování   MeSH
• buněčná smrt MeSH
• genový knockdown metody   veterinární   MeSH
• gonády chemie   MeSH
• komplementární DNA chemie   MeSH
• morfolino * aplikace a dávkování   MeSH
• proteiny vázající RNA analýza   genetika   MeSH
• ryby * genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• sterilizace reprodukční metody   veterinární   MeSH
• zárodečné buňky fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense oligonukleotidy * MeSH
• komplementární DNA MeSH
• morfolino * MeSH
• proteiny vázající RNA 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
• antisense oligonukleotidy * terapeutické užití   genetika   MeSH
• genetické nemoci vrozené genetika   terapie   MeSH
• lidé MeSH
• morfolino terapeutické užití   genetika   MeSH
• mutace * MeSH
• sestřih RNA * MeSH
• vzácné nemoci * genetika   farmakoterapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense oligonukleotidy * MeSH
• morfolino MeSH

With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography. First of all, the effects of different chromatographic conditions (e.g. pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the retention and separation of phosphorothioate oligonucleotides standards on the amide stationary phase were investigated. A set of model oligonucleotides containing a fully modified 21mer and its typical impurities (shortmers and oligonucleotides with different number of thiophosphate modifications) was used. The results showed that the concentration of the salt in the mobile phase as well as its pH, are the most influential parameters with regard to peak shape and separation. The knowledge gained was applied to the analysis of an unpurified 18mer oligonucleotides, analogues of the drug nusinersen used for the treatment of spinal muscular atrophy. The successful separation and identification of twenty-six and twenty-eight impurities was performed with the developed HILIC method. The method was applied to analysis of nusinersen metabolites of serum samples of patients treated with Spinraza.

• Klíčová slova
• Amide stationary phase, Hydrophilic interaction liquid chromatography, Impurities and metabolites, Nusinersen, Phosphorothioate oligonucleotides,
• MeSH
• antisense oligonukleotidy * MeSH
• chromatografie kapalinová metody   MeSH
• fosforothioátové oligonukleotidy * MeSH
• hmotnostní spektrometrie metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• indikátory a reagencie MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense oligonukleotidy * MeSH
• fosforothioátové oligonukleotidy * MeSH
• indikátory a reagencie MeSH
• nusinersen MeSH Prohlížeč

Specific gene knockdown mediated by the antisense oligodeoxynucleotides (AODNs) strategy recently emerged as a rapid and effective tool for probing gene role in plant cells, particularly tip-growing pollen tubes. Here, we describe the protocol for the successful employment of AODN technique in growing tobacco pollen tubes, covering AODN design, application, and analysis of the results. We also discuss the advantages and drawbacks of this method.

• MeSH
• antisense oligonukleotidy genetika   MeSH
• fenotyp MeSH
• genový knockdown * MeSH
• pylová láčka genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• tabák genetika   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense oligonukleotidy MeSH

The effect of oligodeoxyribonucleotides complementary to the region of the so-called pathogenicity domain (nucleotides 42-78) of the upper RNA strand of potato spindle tuber viroid (PSTVd) (severe) on viroid infection was investigated. The oligonucleotides were allowed to form hybrids with PSTVd in the infection mixtures before inoculation. Infectivity tests were performed using intact plants and plant protoplasts. It was found that the DNA oligonucleotides caused significant reduction of viroid infection at plant and single cell levels. The 200-fold molar excess of antisense DNA over viroid RNA is usually sufficient for the complete blocking of viroid infection. The inhibitory effect is strongly sequence specific. Inhibition by corresponding antisense RNA was much less efficient than that caused by antisense DNA.

• MeSH
• antisense DNA farmakologie   MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin mikrobiologie   MeSH
• oligonukleotidy MeSH
• sekvence nukleotidů MeSH
• Solanum tuberosum mikrobiologie   MeSH
• viroidy účinky léků   genetika   MeSH
• virové nemoci prevence a kontrola   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense DNA MeSH
• oligonukleotidy MeSH

BACKGROUND: The genetic and epigenetic alterations observed in acute myeloid leukemia (AML) contribute to its heterogeneity, influencing disease progression response to therapy, and patient outcomes. The use of antisense oligonucleotides (ASOs) technology allows for the design of oligonucleotide inhibitors based on gene sequence information alone, enabling precise targeting of key molecular pathways or specific genes implicated in AML. METHODS AND RESULTS: Midostaurin, a FLT3 specific inhibitor and ASOs targeting particular genes, exons, or mutations was conducted using AML models. This ASOs treatment was designed to bind to exon 7 of the MBNL1 (muscleblind-like) gene. Another target was the FLT3 gene, focusing on two aspects: (a) FLT3-ITD (internal tandem duplication), to inhibit the expression of this aberrant gene form, and (b) the FLT3 in general. Treated and untreated cells were analyzed using quantitative PCR (qPCR), dot blot, and Raman spectroscopy. This study contrasts midostaurin with ASOs that inhibit FLT3 protein production or its isoforms via mRNA degradation. A trend of increased FLT3 expression was observed in midostaurin-treated cells, while ASO-treated cells showed decreased expression, though these changes were not statistically significant. CONCLUSIONS: In AML, exon 7 of MBNL1 is involved in several cellular processes and in this study, exon 7 of MBNL1 was targeted for method optimization, with the highest block of the exon 7 gene variant observed 48 h post-transfection. Midostaurin, a multitargeted kinase inhibitor, acts against the receptor tyrosine kinase FLT3, a critical molecule in AML pathogenesis. While midostaurin blocks FLT3 signaling pathways, it paradoxically increases FLT3 expression.

• Klíčová slova
• Acute myeloid leukemia, Antisense oligonucleotides, FLT3, MBNL1, Target specific therapy,
• MeSH
• akutní myeloidní leukemie * genetika   farmakoterapie   MeSH
• antisense oligonukleotidy * farmakologie   genetika   MeSH
• exony genetika   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• regulace genové exprese u leukemie účinky léků   MeSH
• staurosporin * analogy a deriváty   farmakologie   MeSH
• tyrosinkinasa 3 podobná fms * genetika   antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense oligonukleotidy * MeSH
• FLT3 protein, human MeSH Prohlížeč
• MBNL1 protein, human MeSH Prohlížeč
• midostaurin MeSH Prohlížeč
• proteiny vázající RNA MeSH
• staurosporin * MeSH
• tyrosinkinasa 3 podobná fms * 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.

• MeSH
• antisense oligonukleotidy chemie   metabolismus   MeSH
• buňky 3T3 MeSH
• fluorescenční spektrometrie metody   MeSH
• myši MeSH
• porfyriny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense oligonukleotidy MeSH
• porfyriny MeSH

Several types of isopolar modified oligothymidylates and oligoadenylates (15 mers) with the phosphonate -O-P-CH2-O- internucleotide linkage were prepared. The modified oligonucleotides were subjected to the study of their hybridization properties, resistance against nucleases, and the ability to elicit RNase H activity.

• MeSH
• adenosinmonofosfát chemie   MeSH
• antisense oligonukleotidy chemická syntéza   chemie   metabolismus   MeSH
• hybridizace nukleových kyselin MeSH
• organofosfonáty chemie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• ribonukleasa H metabolismus   MeSH
• thymidinmonofosfát chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosinmonofosfát MeSH
• antisense oligonukleotidy MeSH
• organofosfonáty MeSH
• reagencia zkříženě vázaná MeSH
• ribonukleasa H MeSH
• thymidinmonofosfát MeSH

Interaction, i.e., cellular uptake and intracellular distribution, of synthetic modified antisense oligonucleotide with the B16 melanoma cell line was studied using cationic polyene antibiotic, amphotericin B 3-dimethylaminopropyl amide, as a carrier vector. The antisense oligonucleotide--dT(15) oligomer analogue containing isopolar, nonisosteric, phosphonate-based internucleotide linkages 3'-O-P-CH(2)-O-5'--was labeled with fluorescent tetramethylrhodamine marker. The oligonucleotide itinerancy across the cell membrane and its distribution inside the cell was visualized using fluorescence microimaging. During the first several hours a strong preference staining of the cell nucleus was found. Fluorescence lifetime measurements from the intracellular environment (confocal laser microspectrofluorimeter, frequency domain phase/modulation technique in 1 to 200 MHz frequency region) yielded two spectral components of 4.9 and 1.4 ns lifetime, respectively. While the former component correlates with the previously characterized effect of the fluorophore binding to biomolecular targets in membranes and/or cytoplasm, the latter component is newly observed and its possible origin is discussed.

• MeSH
• amfotericin B farmakologie   MeSH
• antisense oligonukleotidy farmakokinetika   MeSH
• buněčná membrána metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• fluorescenční spektrometrie metody   MeSH
• kationty MeSH
• konfokální mikroskopie MeSH
• lasery MeSH
• melanom experimentální MeSH
• myši MeSH
• oligonukleotidy farmakokinetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amfotericin B MeSH
• amphotericin B3-(N'-dimethylamino)propylamide MeSH Prohlížeč
• antisense oligonukleotidy MeSH
• kationty MeSH
• oligonukleotidy MeSH