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Genes Jul 2023Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile...
Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile epileptic spasms syndrome. The diagnostic criteria of AIC were revised in 2005 to include additional phenotypes that are frequently observed in this patient group. AIC has been traditionally considered as X-linked and male lethal because it almost exclusively affects females. Despite numerous genetic and genomic investigations on AIC, a unifying X-linked cause has not been identified. Here, we performed exome and genome sequencing of 10 females with AIC or suspected AIC based on current criteria. We identified a unique de novo variant, each in different genes: , , , and , in five of these females. Notably, genomic analyses of coding and non-coding single nucleotide variants, short tandem repeats and structural variation highlighted a distinct lack of X-linked candidate genes. We assessed the likely pathogenicity of our candidate autosomal variants using the TOPflash assay for WNT8B and morpholino knockdown in zebrafish () embryos for other candidates. We show expression of and are restricted to clinically relevant cortical tissues during mouse development. Our findings suggest that AIC is genetically heterogeneous with implicated genes converging on molecular pathways central to cortical development.
Topics: Male; Female; Animals; Mice; Aicardi Syndrome; Zebrafish; Chromosome Mapping; Genes, X-Linked; Biological Assay
PubMed: 37628618
DOI: 10.3390/genes14081565 -
Antimicrobial Agents and Chemotherapy Sep 2023Development of new therapeutics against antibiotic resistant pathogenic bacteria is recognized as a priority across the globe. We have reported using peptide-conjugated...
Development of new therapeutics against antibiotic resistant pathogenic bacteria is recognized as a priority across the globe. We have reported using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) as species-specific antibiotics. The oligo sequences, 11 bases are designed to be complementary to specific essential genes near the Shine-Dalgarno site and inhibit translation. Here, we analyzed target specificity and the impact of genetic mutations on lead PPMOs targeting the or gene of . Mutants in PAO1 were generated with four, two, or one base-pair mutations within the 11-base target sequence of the gene. All mutants exhibited increased MICs compared to wild-type PAO1 when treated with the RpsJ PPMO, and the increase in the MICs was proportional to the number of base-pair mutations. Among single base-pair mutants, mutations in the middle of the sequence were more impactful than mutations in 5' or 3' end of the sequence. The increased MICs shown by the mutants could be reversed by PPMOs designed to target the mutated sequence. BALB/c mice infected intratracheally with mutants demonstrated increased lung burden when treated with RpsJ PPMO compared to wild-type PAO1-infected mice treated with RpsJ PPMO. Treating mice with a PPMOs designed to specifically target the mutant sequence was more effective against these mutant strains. These experiments confirm target specificity of two lead PPMOs and illustrate one potential mechanism of resistance that could emerge from an antisense approach.
Topics: Animals; Mice; Morpholinos; Pseudomonas aeruginosa; Anti-Bacterial Agents; Genes, Essential; Mice, Inbred BALB C
PubMed: 37610213
DOI: 10.1128/aac.00245-23 -
STAR Protocols Sep 2023Here, we present a protocol for constructing an ultrasensitive biosensor for exosomal-miRNA detection. We describe steps for preparing graphene quantum...
Here, we present a protocol for constructing an ultrasensitive biosensor for exosomal-miRNA detection. We describe steps for preparing graphene quantum dot-phosphorodiamidate morpholino oligomer hybrids, depositing them onto the reduced graphene oxide field surface, hybridizing analyte miRNA with the sensor probe, and capturing and calculating electrical signals. We also detail procedures for optimizing biosensor construction and evaluating performance. By quantifying plasma exosomal miRNA21, this protocol can identify cancer patients from healthy individuals. For complete details on the use and execution of this protocol, please refer to Li et al..
Topics: Humans; MicroRNAs; Biosensing Techniques
PubMed: 37590152
DOI: 10.1016/j.xpro.2023.102516 -
Cells Aug 2023Cardiovascular diseases (CVDs) are the prevalent cause of mortality worldwide. A combination of environmental and genetic effectors modulates the risk of developing...
Cardiovascular diseases (CVDs) are the prevalent cause of mortality worldwide. A combination of environmental and genetic effectors modulates the risk of developing them. Thus, it is vital to identify candidate genes and elucidate their role in the manifestation of the disease. Large-scale human studies have revealed the implication of Craniofacial Development Protein 1 (CFDP1) in Coronary Artery Disease (CAD). CFDP1 belongs to the evolutionary conserved Bucentaur (BCNT) family, and to date, its function and mechanism of action in Cardiovascular Development are still unclear. We utilized zebrafish to investigate the role of in the developing heart due to the high genomic homology, similarity in heart physiology, and ease of experimental manipulations. We showed that was expressed during development, and we tested two morpholinos and generated a mutant line. The embryos developed arrhythmic hearts and exhibited defective cardiac performance, which led to a lethal phenotype. Findings from both knockdown and knockout experiments showed that abrogation of leads to downregulation of Wnt signaling in embryonic hearts during valve development but without affecting Notch activation in this process. The zebrafish mutant line provides a valuable tool for unveiling the novel mechanism of regulating cardiac physiology and function. is essential for cardiac development, a previously unreported phenotype most likely due to early lethality in mice. The detected phenotype of bradycardia and arrhythmias is an observation with potential clinical relevance for humans carrying heterozygous CFDP1 mutations and their risk of developing CAD.
Topics: Animals; Humans; Cardiovascular Diseases; Heart; Nuclear Proteins; Phenotype; Wnt Signaling Pathway; Zebrafish
PubMed: 37566073
DOI: 10.3390/cells12151994 -
Journal of Controlled Release :... Sep 2023Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood-brain...
Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood-brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ß-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (Aβ) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, L-configured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with non-fluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.
Topics: Mice; Animals; Amyloid beta-Peptides; Alzheimer Disease; Nucleic Acids; Brain; Blood-Brain Barrier; Nanoconjugates; MicroRNAs; Neurons; Disease Models, Animal; Mice, Transgenic
PubMed: 37544515
DOI: 10.1016/j.jconrel.2023.08.001 -
Communications Biology Aug 2023Investigating gene function relies on the efficient manipulation of endogenous gene expression. Currently, a limited number of tools are available to robustly manipulate...
Investigating gene function relies on the efficient manipulation of endogenous gene expression. Currently, a limited number of tools are available to robustly manipulate endogenous gene expression between "on" and "off" states. In this study, we insert a 63 bp coding sequence of T3H38 ribozyme into the 3' untranslated region (UTR) of C. elegans endogenous genes using the CRISPR/Cas9 technology, which reduces the endogenous gene expression to a nearly undetectable level and generated loss-of-function phenotypes similar to that of the genetic null animals. To achieve conditional knockout, a cassette of loxP-flanked transcriptional termination signal and ribozyme is inserted into the 3' UTR of endogenous genes, which eliminates gene expression spatially or temporally via the controllable expression of the Cre recombinase. Conditional endogenous gene turn-on can be achieved by either injecting morpholino, which blocks the ribozyme self-cleavage activity or using the Cre recombinase to remove the loxP-flanked ribozyme. Together, our results demonstrate that these ribozyme-based tools can efficiently manipulate endogenous gene expression both in space and time and expand the toolkit for studying the functions of endogenous genes.
Topics: Animals; RNA, Catalytic; Caenorhabditis elegans; Gene Expression
PubMed: 37542105
DOI: 10.1038/s42003-023-05184-4 -
Scientific Reports Aug 2023Phosphorodiamidate morpholino oligonucleotides (PMOs) are a promising type of antisense oligonucleotides, but their challenging synthesis makes them difficult to access....
Phosphorodiamidate morpholino oligonucleotides (PMOs) are a promising type of antisense oligonucleotides, but their challenging synthesis makes them difficult to access. This research presents an efficient synthetic approach for PMOs using the H-phosphonate approach. The use of phosphonium-type condensing reagents significantly reduced coupling times compared with the current synthetic approach. Furthermore, phosphonium-type condensing reagents facilitated the fragment condensation of PMO, synthesizing up to 8-mer containing all four nucleobases with remarkable coupling efficacy. This is the first report on the convergent synthesis of PMOs. This approach would facilitate the large-scale synthesis of PMOs and accelerate their popularity and accessibility as a next-generation therapy.
Topics: Morpholinos; Organophosphonates; Oligonucleotides, Antisense
PubMed: 37537221
DOI: 10.1038/s41598-023-38698-2 -
Science Translational Medicine Aug 2023Although alternative splicing (AS) drives transcriptional responses and cellular adaptation to environmental stresses, its contributions in organ transplantation have...
Although alternative splicing (AS) drives transcriptional responses and cellular adaptation to environmental stresses, its contributions in organ transplantation have not been appreciated. We have shown that carcinoembryonic antigen-related cell adhesion molecule (Ceacam1; ), a transmembrane biliary glycoprotein expressed in epithelial, endothelial, and immune cells, determines donor liver transplant quality. Here, we studied how AS of affects ischemia-reperfusion injury (IRI) in mouse and human livers. We found that the short cytoplasmic isoform increased during early acute and late resolution phases of warm IRI injury in mice. Transfection of Ceacam1-deficient mouse hepatocytes with adenoviral Ceacam1-S mitigated hypoxia-induced loss of cellular adhesion by repressing the Ask1/p-p38 cell death pathway. Nucleic acid-blocking morpholinos, designed to selectively induce Ceacam1-S, protected hepatocyte cultures against temperature-induced stress in vitro. Luciferase and chromatin immunoprecipitation assays identified direct binding of hypoxia-inducible factor-1α (Hif-1α) to the mouse polypyrimidine tract binding protein 1 () promoter region. Dimethyloxalylglycine protected mouse livers from warm IR stress and hepatocellular damage by inhibiting prolyl hydroxylase domain-containing protein 1 and promoting AS of . Last, analysis of 46 human donor liver grafts revealed that positively correlated with pretransplant expression. This also correlated with better transplant outcomes, including reduced , total bilirubin, proinflammatory , cytokines, immune activation markers , and incidence of delayed complications from biliary anastomosis. This translational study identified mouse Hif-1α-controlled AS of , through transcriptional regulation of promoter region, as a functional underpinning of hepatoprotection against IR stress and tissue damage in liver transplantation.
Topics: Humans; Mice; Animals; Alternative Splicing; Liver Transplantation; Hypoxia-Inducible Factor 1, alpha Subunit; Living Donors; Liver Diseases; Cell Adhesion Molecules; Ischemia
PubMed: 37531413
DOI: 10.1126/scitranslmed.adf2059 -
Pharmaceutics Jun 2023Antisense oligonucleotide (ASO)-mediated exon skipping has become a valuable tool for investigating gene function and developing gene therapy. Machine-learning-based...
Antisense oligonucleotide (ASO)-mediated exon skipping has become a valuable tool for investigating gene function and developing gene therapy. Machine-learning-based computational methods, such as eSkip-Finder, have been developed to predict the efficacy of ASOs via exon skipping. However, these methods are computationally demanding, and the accuracy of predictions remains suboptimal. In this study, we propose a new approach to reduce the computational burden and improve the prediction performance by using feature selection within machine-learning algorithms and ensemble-learning techniques. We evaluated our approach using a dataset of experimentally validated exon-skipping events, dividing it into training and testing sets. Our results demonstrate that using a three-way-voting approach with random forest, gradient boosting, and XGBoost can significantly reduce the computation time to under ten seconds while improving prediction performance, as measured by for both 2'-O-methyl nucleotides (2OMe) and phosphorodiamidate morpholino oligomers (PMOs). Additionally, the feature importance ranking derived from our approach is in good agreement with previously published results. Our findings suggest that our approach has the potential to enhance the accuracy and efficiency of predicting ASO efficacy via exon skipping. It could also facilitate the development of novel therapeutic strategies. This study could contribute to the ongoing efforts to improve ASO design and optimize gene therapy approaches.
PubMed: 37513994
DOI: 10.3390/pharmaceutics15071808 -
Molecules (Basel, Switzerland) Jul 2023Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a... (Review)
Review
Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a neutral phosphorodiamidate linkage. PMOs work via a steric blocking mechanism and are stable towards nucleases' inside cells. PMOs are usually synthesized using phosphoramidate P(V) chemistry. In this review, we will discuss the synthesis of PMOs, phosphoroamidate morpholinos (MO), and thiophosphoramidate morpholinos (TMO).
Topics: Morpholinos; Organophosphorus Compounds; Oligonucleotides, Antisense
PubMed: 37513252
DOI: 10.3390/molecules28145380