RESUMEN

Severe toxic effects of arsenic on human physiology have been of immense concern worldwide. Arsenic causes irrevocable structural and functional disruption of tissues, leading to major diseases in chronically exposed individuals. However, it is yet to be resolved whether the effects result from direct deposition and persistence of arsenic in tissues, or via activation of indirect signaling components. Emerging evidences suggest that gut inhabitants play an active role in orchestrating various aspects of brain physiology, as the gut-brain axis maintains cognitive health, emotions, learning and memory skills. Arsenic-induced dysbiosis may consequentially evoke neurotoxicity, eventually leading to anxiety and depression. To delineate the mechanism of action, mice were exposed to different concentrations of arsenic. Enrichment of Gram-negative bacteria and compromised barrier integrity of the gut enhanced lipopolysaccharide (LPS) level in the bloodstream, which in turn elicited systemic inflammation. Subsequent alterations in neurotransmitter levels, microglial activation and histoarchitectural disruption in brain triggered onset of anxiety- and depression-like behaviour in a dose-dependent manner. Finally, to confirm whether the neurotoxic effects are specifically a consequence of modulation of gut microbiota (GM) by arsenic and not arsenic accumulation in the brain, fecal microbiota transplantations (FMT) were performed from arsenic-exposed mice to healthy recipients. 16S rRNA gene sequencing indicated major alterations in GM population in FMT mice, leading to severe structural, functional and behavioural alterations. Moreover, suppression of Toll-like receptor 4 (TLR4) using vivo-morpholino oligomers (VMO) indicated restoration of the altered parameters towards normalcy in FMT mice, confirming direct involvement of the GM in inducing neurotoxicity through the arsenic-gut-brain axis. This study accentuates the potential role of the gut microbiota in promoting neurotoxicity in arsenic-exposed mice, and has immense relevance in predicting neurotoxicity under altered conditions of the gut for designing therapeutic interventions that will target gut dysbiosis to attenuate arsenic-mediated neurotoxicity.

RESUMEN

Multispecific T-cell-engaging scaffolds have emerged as effective anticancer therapies for the treatment of hematological malignancies. Approaches that modulate cancer cell targeting and provide personalized, multispecific immunotherapeutics are needed. Here, we report on a modular, split antibody-like approach consisting of Fab' fragments modified with complementary morpholino oligonucleotides (MORFs). We synthesized a library of B-cell-targeting Fab'-MORF1 conjugates that self-assemble, via a Watson-Crick base pairing hybridization, with a complementary T-cell-engaging Fab'-MORF2 conjugate. We aptly titled our technology multiantigen T-cell hybridizers (MATCH). Using MATCH, cancer-specific T-cell recruitment was achieved utilizing four B-cell antigen targets: CD20, CD38, BCMA, and SLAMF7. The antigen expression profiles of various malignant B-cell lines were produced, and using these distinct profiles, cell-specific T-cell activation was attained on lymphoma, leukemia, and multiple myeloma cell lines in vitro. T-cell rechallenge experiments demonstrated the modular approach of MATCH by sequentially activating the same T-cell cohort against three different cancers using cancer antigen-specific Fab'-MORF1 conjugates. Furthermore, MATCH's efficacy was demonstrated in vivo by treating xenograft mouse models of human non-Hodgkin's lymphoma with CD20-directed MATCH therapy. In the pilot study, a single dose of MATCH allowed for long-term survival of all treated mice compared to saline control. In a second in vivo model, insights regarding optimal T-cell-to-target cell ratio were gleaned when a ratio of 5:1 T-cell-to-target cell MATCH-treated mice significantly delayed the onset of disease compared to higher and lower ratios.

Asunto(s)

RESUMEN

Sequencing of phosphorodiamidate morpholino oligomers (PMOs) by hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) is reported. The MS/MS analysis was performed using a quadrupole/time-of-flight (Q-ToF) mass analyzer and collision induced dissociation (CID) in negative ion mode. To improve MS sensitivity in negative ion mode, HILIC conditions, including the separation column, mobile phases, and MS parameters, were optimized. Using the developed HILIC-CID-MS/MS method, 100% sequence coverage was achieved for PMOs ranging from 18-mer to 25-mer. Additionally, the method was successfully applied to identifying positional isomers of n - 1 deletion impurities present in PMO drug substances.

RESUMEN

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

RESUMEN

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Asunto(s)

RESUMEN

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 (SMN1) gene, hindering the production of functional survival motor neuron (SMN) proteins. Antisense oligonucleotides (ASOs), a versatile DNA-like drug, are adept at binding to target RNA to prevent translation or promote alternative splicing. Nusinersen is an FDA-approved ASO for the treatment of SMA. It effectively promotes alternative splicing in pre-mRNA transcribed from the SMN2 gene, an analog of the SMN1 gene, to produce a greater amount of full-length SMN protein, to compensate for the loss of functional protein translated from SMN1. Despite its efficacy in ameliorating SMA symptoms, the cellular uptake of these ASOs is suboptimal, and their inability to penetrate the CNS necessitates invasive lumbar punctures. Cell-penetrating peptides (CPPs), which can be conjugated to ASOs, represent a promising approach to improve the efficiency of these treatments for SMA and have the potential to transverse the blood-brain barrier to circumvent the need for intrusive intrathecal injections and their associated adverse effects. This review provides a comprehensive analysis of ASO therapies, their application for the treatment of SMA, and the encouraging potential of CPPs as delivery systems to improve ASO uptake and overall efficiency.

Asunto(s)

RESUMEN

Zebrafish larvae have emerged as a valuable model for studying heart physiology and pathophysiology, as well as for drug discovery, in part thanks to its transparency, which simplifies microscopy. However, in fluorescence-based optical mapping, the beating of the heart results in motion artifacts. Two approaches have been employed to eliminate heart motion during calcium or voltage mapping in zebrafish larvae: the knockdown of cardiac troponin T2A and the use of myosin inhibitors. However, these methods disrupt the mechano-electric and mechano-mechanic coupling mechanisms. We have used ratiometric genetically encoded biosensors to image calcium in the beating heart of intact zebrafish larvae because ratiometric quantification corrects for motion artifacts. In this study, we found that halting heart motion by genetic means (injection of tnnt2a morpholino) or chemical tools (incubation with para-aminoblebbistatin) leads to bradycardia, and increases calcium levels and the size of the calcium transients, likely by abolishing a feedback mechanism that connects contraction with calcium regulation. These outcomes were not influenced by the calcium-binding domain of the gene-encoded biosensors employed, as biosensors with a modified troponin C (Twitch-4), calmodulin (mCyRFP1-GCaMP6f), or the photoprotein aequorin (GFP-aequorin) all yielded similar results. Cardiac contraction appears to be an important regulator of systolic and diastolic Ca2+ levels, and of the heart rate.

Asunto(s)

RESUMEN

A thorough search for the development of innovative drugs to treat tuberculosis, especially considering the urgent need to address developing drug resistance, we report here a synthetic series of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o) as potent anti-tubercular agents. These morpholino-indolizines were synthesized by reacting 4-morpholino pyridinium salts, with various electron-deficient acetylenes to afford the ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o). All synthesized intermediate and final compounds are characterized by spectroscopic methods such as 1H NMR, 13C NMR and HRMS and further examined for their anti-tubercular activity against the M. tuberculosis H37Rv strain (ATCC 27294-American type cell culture). All the compounds screened for anti-tubercular activity in the range of 6.25-50 µM against the H37Rv strain of Mycobacterium tuberculosis. Compound 5g showed prominent activity with MIC99 2.55 µg/mL whereas compounds 5d and 5j showed activity with MIC99 18.91 µg/mL and 25.07 µg/mL, respectively. In silico analysis of these compounds revealed drug-likeness. Additionally, the molecular target identification for Malate synthase (PDB 5CBB) is attained by computational approach. The compound 5g with a MIC99 value of 2.55 µg/mL against M. tuberculosis H37Rv emerged as the most promising anti-TB drug and in silico investigations suggest Malate synthase (5CBB) might be the compound's possible target.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.

Asunto(s)

RESUMEN

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule-induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using azido-caged nucleobases that are site-specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, efficiently blocks Watson-Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule-induced gene knockdown in mammalian cells, zebrafish embryos, and frog embryos. We validated the general applicability of this approach by targeting three different genes.

Asunto(s)

RESUMEN

Introduction. Influenza is a global health issue causing substantial health and economic burdens on affected populations. Routine, annual vaccination for influenza virus is recommended for all persons older than 6 months of age. The propagation of the influenza virus for vaccine production is predominantly through embryonated chicken eggs.Hypothesis/Gap Statement. Many challenges face the propagation of the virus, including but not limited to low yields and lengthy production times. The development of a method to increase vaccine production in eggs or cell lines by suppressing cellular gene expression would be helpful to overcome some of the challenges facing influenza vaccine production.Aims. This study aimed to increase influenza virus titres by using a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an antisense molecule, to suppress protein expression of the host genes interferon alpha (IFN-α) and interferon beta (IFN-ß) in chicken embryo fibroblast (DF-1) cells.Methods. The toxicity of PPMOs was evaluated by cytotoxicity assays, and their specificity to inhibit IFN-α and IFN-ß proteins was measured by ELISA. We evaluated the potential of anti-IFN-α and anti-IFN-ß PPMOs to reduce the antiviral proteins in influenza virus-infected DF-1 cells and compared the virus titres to untreated controls, nonsense-PPMO and JAK/STAT inhibitors. The effects of complementation and reconstitution of IFN-α and IFN-ß proteins in PPMO-treated-infected cells were evaluated, and the virus titres were compared between treatment groups.Results. Suppression of IFN-α by PPMO resulted in significantly reduced levels of IFN-α protein in treated wells, as measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMOs increased the ability of the influenza virus to replicate in DF-1 cells. Over a 2-log10 increase in viral production was observed in anti-IFN-α and IFN-ß PPMO-treated wells compared to those of untreated controls at the initial viral input of 0.1 multiplicity of infection. The data from complementation and reconstitution of IFN-α and IFN-ß proteins in PPMO-treated-infected cells was about 82 and 97% compared to the combined PPMO-treated but uncomplemented group and untreated group, respectively. There was a 0.5-log10 increase in virus titre when treated with anti-IFN-α and IFN-ß PPMO compared to virus titre when treated with JAK/STAT inhibitors.Conclusions. This study emphasizes the utility of PPMO in allowing cell cultures to produce increased levels of influenza for vaccine production or alternatively, as a screening tool to cheaply test targets prior to the development of permanent knockouts of host gene expression.

Asunto(s)

RESUMEN

Pathogenic variants in the DES gene clinically manifest as progressive skeletal muscle weakness, cardiomyopathy with associated severe arrhythmias, and respiratory insufficiency, and are collectively known as desminopathies. While most DES pathogenic variants act via a dominant mechanism, recessively acting variants have also been reported. Currently, there are no effective therapeutic interventions for desminopathies of any type. Here, we report an affected individual with rapidly progressive dilated cardiomyopathy, requiring heart transplantation at age 13 years, in the setting of childhood-onset skeletal muscle weakness. We identified biallelic DES variants (c.640-13 T>A and c.1288+1 G>A) and show aberrant DES gene splicing in the affected individual's muscle. Through the generation of an inducible lentiviral system, we transdifferentiated fibroblast cultures derived from the affected individual into myoblasts and validated this system using RNA sequencing. We tested rationally designed, custom antisense oligonucleotides to screen for splice correction in these transdifferentiated cells and a functional minigene splicing assay. However, rather than correctly redirecting splicing, we found them to induce undesired exon skipping. Our results indicate that, while an individual precision-based molecular therapeutic approach to splice-altering pathogenic variants is promising, careful preclinical testing is imperative for each novel variant to test the feasibility of this type of approach for translation.

Asunto(s)

RESUMEN

DprE1 is involved in the synthesis of Mycobacterium tuberculosis cell wall and is a potent drug target for Tuberculosis (TB) treatment. The structure and dynamics of the loops L-I and L-II flanking the inhibitor binding site was studied using molecular dynamics (MD) simulation and MMPBSA in Amber v18. Docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) of 55 Morpholino-pyrimidine (MP) inhibitors was carried out using Autodock v1.2.0 and Forge v10. ADMET analysis was done using SwissADME and pkCSM. All MP inhibitors docked in the DprE1 binding pocket, making contacts with L-II residues. MD studies showed that L-I and L-II unfold in the absence of the inhibitor but fold stably structure with reduced protein motions in the presence of MP-38, the highest affinity inhibitor. This was confirmed by k-means clustering and secondary structure analysis. L-II residues, L317, F320 and R325 contributed most towards the MMPBSA binding free energy of MP-38. A robust field-based 3D-QSAR model showed values of r2train = 0.982, r2test = 0.702 and q2 = 0.516. The MP inhibitor field points were broadly divided into negative electrostatics near the A, B rings and hydrophobic electrostatics near the D, E rings. Addition of negative groups at methanone position and ring B as well as addition of hydrophobic and bulky groups at ring E will improve activity. Highly active compounds 47, 49 and 50 of MP series exhibited highly favourable drug-like properties. SAR and ADMET insights attained from this model will help in the development of active DprE1 inhibitors in future.Communicated by Ramaswamy H. Sarma.

RESUMEN

BACKGROUND: The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration of functional dystrophin levels is a fundamental approach for DMD therapy. Electrical impedance myography (EIM) is an emerging tool that provides noninvasive monitoring of muscle conditions and has been suggested as a treatment response biomarker in diverse indications. Although magnetic resonance imaging (MRI) of skeletal muscles has become a standard measurement in clinical trials for DMD, EIM offers distinct advantages, such as portability, user-friendliness, and reduced cost, allowing for remote monitoring of disease progression or response to therapy. To investigate the potential of EIM as a biomarker for DMD, we compared longitudinal EIM data with MRI/histopathological data from an X-linked muscular dystrophy (mdx) mouse model of DMD. In addition, we investigated whether EIM could detect dystrophin-related changes in muscles using antisense-mediated exon skipping in mdx mice. METHODS: The MRI data for muscle T2, the magnetic resonance spectroscopy (MRS) data for fat fraction, and three EIM parameters with histopathology were longitudinally obtained from the hindlimb muscles of wild-type (WT) and mdx mice. In the EIM study, a cell-penetrating peptide (Pip9b2) conjugated antisense phosphorodiamidate morpholino oligomer (PPMO), designed to induce exon-skipping and restore functional dystrophin production, was administered intravenously to mdx mice. RESULTS: MRI imaging in mdx mice showed higher T2 intensity at 6 weeks of age in hindlimb muscles compared to WT mice, which decreased at ≥ 9 weeks of age. In contrast, EIM reactance began to decline at 12 weeks of age, with peak reduction at 18 weeks of age in mdx mice. This decline was associated with myofiber atrophy and connective tissue infiltration in the skeletal muscles. Repeated dosing of PPMO (10 mg/kg, 4 times every 2 weeks) in mdx mice led to an increase in muscular dystrophin protein and reversed the decrease in EIM reactance. CONCLUSIONS: These findings suggest that muscle T2 MRI is sensitive to the early inflammatory response associated with dystrophin deficiency, whereas EIM provides a valuable biomarker for the noninvasive monitoring of subsequent changes in skeletal muscle composition. Furthermore, EIM reactance has the potential to monitor dystrophin-deficient muscle abnormalities and their recovery in response to antisense-mediated exon skipping.

Asunto(s)

RESUMEN

Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two asb5 gene isoforms, asb5a and asb5b. Currently, the effects of asb5 gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated asb5a-knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar asb5a and asb5b expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target asb5b in wild-type and asb5a-knockout zebrafish. Knocking down asb5b in the wild-type had no phenotypic impact, but simultaneous asb5b knockdown in asb5a-knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in asb5a and asb5b, with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into asb5's mechanism.

Asunto(s)

RESUMEN

In bioprocesses, the pH value is a critical process parameter that requires monitoring and control. For pH monitoring, potentiometric methods such as pH electrodes are state of the art. However, they are invasive and show measurement value drift. Spectroscopic pH monitoring is a non-invasive alternative to potentiometric methods avoiding this measurement value drift. In this study, we developed the Good pH probe, which is an approach for spectroscopic pH monitoring in bioprocesses with an effective working range between pH 6 and pH 8 that does not require the estimation of activity coefficients. The Good pH probe combines for the first time the Good buffer 3-(N-morpholino)propanesulfonic acid (MOPS) as pH indicator with Raman spectroscopy as spectroscopic technique, and Indirect Hard Modeling (IHM) for the spectral evaluation. During a detailed characterization, we proved that the Good pH probe is reversible, exhibits no temperature dependence between 15 and 40 °C, has low sensitivity to the ionic strength up to 1100 mM, and is applicable in more complex systems, in which other components significantly superimpose the spectral features of MOPS. Finally, the Good pH probe was successfully used for non-invasive pH in-line monitoring during an industrially relevant enzyme-catalyzed reaction with a root mean square error of prediction (RMSEP) of 0.04 pH levels. Thus, the Good pH probe extends the list of critical process parameters monitorable using Raman spectroscopy and IHM by the pH value.

RESUMEN

The blood-brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm2. To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture.

RESUMEN

Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising biological and pharmacological properties for antisense applications. Despite their great potential, the efficient delivery of these therapeutic agents to target cells remains a major obstacle to their widespread use. Cellular uptake of naked PMO is poor. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular uptake and intracellular delivery of oligonucleotide-based drugs. Among these, the DG9 peptide has been identified as a versatile CPP with remarkable potential for enhancing the delivery of ASO-based therapeutics due to its unique structural features. Notably, in the context of phosphorodiamidate morpholino oligomers (PMOs), DG9 has shown promise in enhancing delivery while maintaining a favorable toxicity profile. A few studies have highlighted the potential of DG9-conjugated PMOs in DMD (Duchenne Muscular Dystrophy) and SMA (Spinal Muscular Atrophy), displaying significant exon skipping/inclusion and functional improvements in animal models. The article provides an overview of a detailed understanding of the challenges that ASOs face prior to reaching their targets and continued advances in methods to improve their delivery to target sites and cellular uptake, focusing on DG9, which aims to harness ASOs' full potential in precision medicine.

Asunto(s)

RESUMEN

Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated antisense oligonucleotides for exon 44 skipping (applicable to approximately 6% of all Duchenne muscular dystrophy patients) to improve activity by using a novel antisense oligonucleotide design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to target two splicing regulators in exon 44 simultaneously, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a Duchenne muscular dystrophy patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an antisense oligonucleotide with a novel connected-sequence design, showed highly efficient exon skipping both in vitro and in vivo.