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
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
Giardia lamblia, a major parasite, is an emerging model organism due to its compact genomic arrangement and composition. The most popular reverse genetic technique, RNAi, is ineffective in Giardia. In contrast, protein depletion by translation blocking morpholinos is suitable for most gene targets and provides up to 80% depletion of the target protein. The method is fast, reliable, and specific. After antisense morpholino oligomer delivery into Giardia trophozoites by electroporation, the cells can be used for many subsequent analyses 8-48 h after treatment. In this chapter, suitable gene tags, plasmids, and techniques necessary for proper morpholino targeting are described.
- MeSH
- Electroporation MeSH
- Gene Expression MeSH
- Genetic Vectors genetics MeSH
- Gene Knockdown Techniques * MeSH
- Giardia lamblia genetics immunology metabolism MeSH
- Cloning, Molecular MeSH
- Morpholinos administration & dosage chemistry genetics MeSH
- Gene Order MeSH
- Protein Biosynthesis genetics MeSH
- Protozoan Proteins genetics metabolism MeSH
- Recombinant Fusion Proteins MeSH
- Base Sequence MeSH
- Transformation, Genetic MeSH
- Publication type
- Journal Article MeSH
PURPOSE: This study evaluated the efficacy, safety, pharmacodynamics (PD), pharmacokinetics (PK), and immunogenicity of SB16 versus reference denosumab (DEN) up to 18 months in postmenopausal osteoporosis (PMO) patients, and assessed outcomes after switching from DEN to SB16 compared to those who continued with DEN or SB16. METHODS: 457 PMO patients were initially randomized, with 407 re-randomized at Month 12 to either continue DEN (DEN+DEN), switch to SB16 (DEN+SB16), or continue SB16 (SB16 + SB16) through Month 18. Efficacy was assessed by the percent change from baseline in bone mineral density (BMD) at the lumbar spine, total hip, and femoral neck. Safety, PD, PK, and immunogenicity were evaluated throughout the study period. RESULTS: Mean percent changes from baseline in lumbar spine, total hip, and femoral neck BMD at Month 18 were comparable across treatment groups, indicating comparable efficacy between SB16 and DEN. The mean percent change in lumbar spine BMD was 6.8 % (SB16 + SB16), 6.2 % (DEN+SB16), and 6.8 % (DEN+DEN). Total hip BMD increased by 4.4 %, 3.5 %, and 4.0 %, and femoral neck BMD by 3.4 %, 3.1 %, and 2.7 % for SB16 + SB16, DEN+SB16, and DEN+DEN, respectively. Safety profiles were similar among groups, with no new safety concerns identified after switching. Only one patient in the DEN+SB16 group developed non-neutralizing anti-drug antibodies by Month 18, indicating a low immunogenicity risk for SB16. CONCLUSION: Switching from DEN to SB16 demonstrated comparable efficacy, safety, PD, PK, and immunogenicity in PMO patients relative to those who continued DEN. SB16 was well tolerated over 18 months, demonstrating comparable outcomes to DEN.
- MeSH
- Lumbar Vertebrae drug effects MeSH
- Denosumab * therapeutic use MeSH
- Bone Density Conservation Agents * therapeutic use MeSH
- Bone Density * drug effects MeSH
- Middle Aged MeSH
- Humans MeSH
- Osteoporosis, Postmenopausal * drug therapy MeSH
- Aged MeSH
- Treatment Outcome MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Clinical Trial, Phase III MeSH
- Randomized Controlled Trial MeSH
- Comparative Study MeSH
Cleft lip and/or palate (CL/P) are common structural birth defects in humans. We used exome sequencing to study a patient with bilateral CL/P and identified a single nucleotide deletion in the patient and her similarly affected son—c.546_546delG, predicting p.Gln183Argfs*57 in the Distal-less 4 (DLX4) gene. The sequence variant was absent from databases, predicted to be deleterious and was verified by Sanger sequencing. In mammals, there are three Dlx homeobox clusters with closely located gene pairs (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6). In situ hybridization showed that Dlx4 was expressed in the mesenchyme of the murine palatal shelves at E12.5, prior to palate closure. Wild-type human DLX4, but not mutant DLX4_c.546delG, could activate two murine Dlx conserved regulatory elements, implying that the mutation caused haploinsufficiency. We showed that reduced DLX4 expression after short interfering RNA treatment in a human cell line resulted in significant up-regulation of DLX3, DLX5 and DLX6, with reduced expression of DLX2 and significant up-regulation of BMP4, although the increased BMP4 expression was demonstrated only in HeLa cells. We used antisense morpholino oligonucleotides to target the orthologous Danio rerio gene, dlx4b, and found reduced cranial size and abnormal cartilaginous elements. We sequenced DLX4 in 155 patients with non-syndromic CL/P and CP, but observed no sequence variants. From the published literature, Dlx1/Dlx2 double homozygous null mice and Dlx5 homozygous null mice both have clefts of the secondary palate. This first finding of a DLX4 mutation in a family with CL/P establishes DLX4 as a potential cause of human clefts.
- MeSH
- Jaw Abnormalities genetics pathology MeSH
- Zebrafish MeSH
- Exome genetics MeSH
- HeLa Cells MeSH
- Homeodomain Proteins biosynthesis genetics MeSH
- Bone Morphogenetic Protein 4 genetics MeSH
- Humans MeSH
- Mesoderm metabolism MeSH
- Morpholinos MeSH
- Brain abnormalities pathology MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Zebrafish Proteins genetics MeSH
- Cleft Palate genetics pathology MeSH
- Cleft Lip genetics pathology MeSH
- Transcription Factors biosynthesis genetics MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Gene Expression Regulation, Developmental MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, N.I.H., Extramural MeSH
- Keywords
- ataluren, eteplirsen, nusinersen, Translarna, Spinraza, Exondys 51,
- MeSH
- Child MeSH
- Muscular Dystrophy, Duchenne * epidemiology drug therapy physiopathology MeSH
- Humans MeSH
- Morpholinos pharmacology therapeutic use MeSH
- Neuromuscular Diseases * genetics therapy MeSH
- Oligonucleotides pharmacology therapeutic use MeSH
- Oxadiazoles pharmacology therapeutic use MeSH
- Spinal Muscular Atrophies of Childhood * diagnosis drug therapy genetics classification MeSH
- Rare Diseases drug therapy genetics MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Úvod: Existence cirkadiálního rytmu je již dlouhou dobu známa u řady biologických pochodů, jako je např. sekrece hormonů či aktivita autonomního nervového systému. Trvale narůstá také počet důkazů o existenci cirkadiálního rytmu u kardiovaskulárních příhod, zejména u infarktu myokardu, náhlé srdeční smrti a také u cévní mozkové příhody. I když jsou patofyziologie a mechanizmus těchto variací trvale předmětem výzkumu, nejsou zatím dostatečně známé. Srdeční frekvence, krevní tlak a některé neurohumorální vazoaktivní faktory, jako je norepinefrin a renin, vykazují vzestup v ranních hodinách. Cílem naší práce bylo posoudit diurnální variabilitu plazmatických hladin big endotelinu a NT-proBNP u nemocných s těžkým chronickým srdečním selháním. Soubor nemocných: 13 nemocných s těžkým srdečním selháním, stabilní alespoň jeden měsíc, muži/ ženy - 8/ 5, NYHA III/ IV - 11/ 2, průměrná ejekční frakce levé komory 23 +/- 5 %, průměrný kardiotorakální index 59 +/- 7 %, všichni léčeni blokádou RAAS (11krát ACE- I, 2krát ARB), všichni léčeni diuretiky, 12 nemocných léčeno beta-blokátory, 7 digoxinem. Etiologie srdečního selhání - ICHS/ DKMP - 9/ 4. Metodika: Během standardního denního režimu byla odebírána krev na stanovení plazmatické hladiny big endotelinu a NT-proBNP každé 2 hod. Výsledky: Průměrná plazmatická hladina big endotelinu statisticky významně kolísala v rozmezí od 1,25 do 1,71 pmol/ l (norma do 0,7 pmol/ l). Průměrná plazmatická hladina NT-proBNP statisticky nevýznamně kolísala v rozmezí od 782 do 934 pmol/ l (norma do 350 pmo/ l). Závěr: Plazmatická hladina NT-proBNP je stabilní po celých 24 hod a nevykazuje diurnální variabilitu. Plazmatická hladina big endotelinu má ranní vrchol po postupném vzestupu v době klidu. Plazmatickou hladina NT-proBNP je možno odebrat kdykoliv během 24 hod, plazmatická hladina big endotelinu by se měla odebírat za standardních podmínek.1
Introduction: Circadian rhytmus have long been recognized to occur in many biologic phenomena, including secretion of hormones as well as autonomic nervous system. There is increasing evidence that circadian rhythms have been also found in cardiovascular events, for example, myocardial infarction, sudden cardiac death as well as stroke have shown a circadian pattern of the distribution. The pathophysiology and the mechanism underlying these variations are the focus of much investigation, while i tis not full understood up to date. Heart rate, blood pressure, neurohumoral vasoactive factors, such as plasma norepinephrine levels and renin activity, and probably also contractility are increased in the morning hours. The aim of our study was to evaluate the circadian variability of plasma big endothelin and NT‑proBNP level in patients with severe heart failure. Patients: 13 patients with severe heart failure, stable for at least one month, male/ female – 8/ 5, NYHA III/ IV – 11/ 2, mean left ventricle ejection fraction 23 ± 5 %, mean cardiothoracic ratio 59 ± 7 %, all treated with RAAS blocade (11× ACE‑ I, 2× ARB), all treated with diuretics, 12 patients treated with beta‑blockers, 7 with digoxin. The cause of heart failure was ischemic heart disease (9) or dilated cardiomyopathy (4). Methods: Blood samples for big endothelin and NT‑proBNP were taken every two hours during a standartised daily regime. Results: Mean plasma level of big endothelin (ranging from 1.25 to 1.71 pmol/ l) had significant diurnal variability (upper limit of normal values 0.7 pmol/ l). Mean plasma level of NT‑proBNP (ranging from 782 to 934 pmol/ l) had no diurnal variability (upper limit of normal values of 350 pmo/ l). Summary: Plasma level of NT‑proBNP is stable during 24 hours and shows no circadian variability. Plasma big endothelin showed a morning peak after a systematic increase during bed rest. NT‑proBNP could be evaluated any time during the day, big endothelin sample should be taken during standartised condition.
