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Biomedicine & Pharmacotherapy =... Jun 2024Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with...
Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.
Topics: Animals; Blood-Brain Barrier; Oligonucleotides, Antisense; Humans; Apolipoproteins E; Mice, Transgenic; Mice; Morpholinos; Survival of Motor Neuron 2 Protein; Muscular Atrophy, Spinal; Drug Delivery Systems; Fibroblasts; Brain; Peptides; Cell-Penetrating Peptides
PubMed: 38749176
DOI: 10.1016/j.biopha.2024.116737 -
BioRxiv : the Preprint Server For... May 2024Propranolol reduces experimental murine cerebral cavernous malformations (CCMs) and prevents embryonic caudal venous plexus (CVP) lesions in zebrafish that follow mosaic...
Propranolol reduces experimental murine cerebral cavernous malformations (CCMs) and prevents embryonic caudal venous plexus (CVP) lesions in zebrafish that follow mosaic inactivation of . Because morpholino silencing of the β1 adrenergic receptor () prevents the embryonic CVP lesion, we proposed that plays a role in CCM pathogenesis. Here we report that zebrafish exhibited 86% fewer CVP lesions and 87% reduction of CCM lesion volume relative to wild type brood mates at 2dpf and 8-10 weeks stage, respectively. Treatment with metoprolol, a β1 selective antagonist, yielded a similar reduction in CCM lesion volume. zebrafish embryos exhibited reduced heart rate and contractility and reduced CVP blood flow. Similarly, slowing the heart and eliminating the blood flow in CVP by administration of 2,3-BDM suppressed the CVP lesion. In sum, our findings provide genetic and pharmacological evidence that the therapeutic effect of propranolol on CCM is achieved through β1 receptor antagonism.
PubMed: 38746306
DOI: 10.1101/2024.05.05.592554 -
Hereditas Apr 2024Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been...
BACKGROUND
Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants.
RESULTS
Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos.
CONCLUSIONS
Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.
Topics: Animals; Zebrafish; Nicotinamide Phosphoribosyltransferase; Hematopoiesis; Mutation; Zebrafish Proteins; Phenotype; CRISPR-Cas Systems; NAD; Gene Knockdown Techniques; Morpholinos
PubMed: 38685093
DOI: 10.1186/s41065-024-00318-y -
Biomedicines Apr 2024Casimersen (AMONDYS 45) is an antisense oligonucleotide of the phosphorodiamidate morpholino oligomer subclass developed by Sarepta therapeutics. It was approved by the... (Review)
Review
Casimersen (AMONDYS 45) is an antisense oligonucleotide of the phosphorodiamidate morpholino oligomer subclass developed by Sarepta therapeutics. It was approved by the Food and Drug Administration (FDA) in February 2021 to treat Duchenne muscular dystrophy (DMD) in patients whose gene mutation is amenable to exon 45 skipping. Administered intravenously, casimersen binds to the pre-mRNA of the gene to skip a mutated region of an exon, thereby producing an internally truncated yet functional dystrophin protein in DMD patients. This is essential in maintaining the structure of a myocyte membrane. While casimersen is currently continuing in phase III of clinical trials in various countries, it was granted approval by the FDA under the accelerated approval program due to its observed increase in dystrophin production. This article discusses the pathophysiology of DMD, summarizes available treatments thus far, and provides a full drug review of casimersen (AMONDYS 45).
PubMed: 38672266
DOI: 10.3390/biomedicines12040912 -
ELife Apr 2024We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail...
We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism. SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.
Topics: Animals; Cell Movement; Fibroblast Growth Factors; High-Temperature Requirement A Serine Peptidase 1; Neural Crest; Serpin E2; Signal Transduction; Xenopus laevis; Xenopus Proteins
PubMed: 38634469
DOI: 10.7554/eLife.91864 -
Genetics Jun 2024Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially... (Review)
Review
Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.
Topics: Giardia; Giardiasis; Protozoan Proteins; Humans; CRISPR-Cas Systems; Animals
PubMed: 38626297
DOI: 10.1093/genetics/iyae038 -
Scientific Reports Apr 2024The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating...
The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable-and easily accessible-tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.
Topics: Humans; Animals; Mice; Zebrafish; Lateral Line System; Mechanotransduction, Cellular; Hair Cells, Auditory; Hearing Loss; Muscle Proteins
PubMed: 38570547
DOI: 10.1038/s41598-024-58138-z -
Research Square Mar 2024CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone...
CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our In vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.
PubMed: 38562718
DOI: 10.21203/rs.3.rs-4022423/v1 -
Scientific Reports Mar 2024Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1...
Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with K values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.
Topics: Animals; Humans; DNA; G-Quadruplexes; HEK293 Cells; HeLa Cells; Mandibulofacial Dysostosis; Nuclear Proteins; Phosphoproteins; RNA-Binding Proteins; Transcription Factors; Zebrafish
PubMed: 38553547
DOI: 10.1038/s41598-024-58255-9 -
Scientific Reports Mar 2024Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish...
Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish IM strain from the India (IND) strain through full sib-pair mating for 16 generations. However, the IM fish laid a small number of offspring and had a short lifespan, implying the need for discreet care in breeding. Here, we report the subsequent establishment of IM strain as well as the generation of a new inbred zebrafish strain, Mishima-AB (M-AB). M-AB was derived from the *AB strain by full sib-pair mating for over 20 generations, which fulfills the general criterion for the establishment of an inbred strain. In contrast to the IM case, maintenance of the M-AB strain by sib-pair mating required almost no special handling. Genome sequencing of IM individuals from the 47th generation and M-AB individuals from the 27th generation revealed that SNP-based genomic heterogeneity across whole-genome nucleotides was 0.008% and 0.011%, respectively. These percentages were much lower than those of the parental IND (0.197%) and *AB (0.086%) strains. These results indicate that the genomes of these inbred strains were highly homogenous. We also demonstrated the successful microinjection of antisense morpholinos, CRISPR/Cas9, and foreign genes into M-AB embryos at the 1-cell stage. Overall, we report the establishment of a zebrafish inbred strain, M-AB, which is capable of regular breeding and genetic manipulation. This strain will be useful for the analysis of genetically susceptible phenotypes such as behaviors, microbiome features and drug susceptibility.
Topics: Animals; Zebrafish; Inbreeding; Genome; Chromosome Mapping; Phenotype
PubMed: 38548817
DOI: 10.1038/s41598-024-57699-3