ID: 32093728
Title: Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD.
Abstract: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

ID: 34723509
Title: A Microfluidics-Based Scalable Approach to Generate Extracellular Vesicles with Enhanced Therapeutic MicroRNA Loading for Intranasal Delivery to Mouse Glioblastomas.
Abstract: Extracellular vesicles (EVs), including exosomes and microvesicles derived from different cell sources, are used as promising nanovesicles for delivering therapeutic microRNAs (miRNAs) and drugs in cancer therapy. However, their clinical translation is limited by the quantity, size heterogeneity, and drug or small RNA loading efficiency. Herein, we developed a scalable microfluidic platform that can load therapeutic miRNAs (antimiRNA-21 and miRNA-100) and drugs while controlling the size of microfluidically processed EVs (mpEVs) using a pressure-based disruption and reconstitution process. We prepared mpEVs of optimal size using microvesicles isolated from neural stem cells engineered to overexpress CXCR4 receptor and characterized them for charge and miRNA loading efficiency. Since the delivery of therapeutic miRNAs to brain cancer is limited by the blood-brain barrier (BBB), we adopted intranasal administration of miRNA-loaded CXCR4-engineered mpEVs in orthotopic GBM mouse models and observed a consistent pattern of mpEVs trafficking across the nasal epithelia, bypassing the BBB into the intracranial compartment. In addition, the CXCR4-engineered mpEVs manifested selective tropism toward GBMs by stromal-derived factor-1 chemotaxis to deliver their miRNA cargo. The delivered miRNAs sensitized GBM cells to temozolomide, resulting in prominent tumor regression, and improved the overall survival of mice. A simple and efficient approach of packaging miRNAs in mpEVs using microfluidics, combined with a noninvasive nose-to-brain delivery route presents far-reaching potential opportunities to improve GBM therapy in clinical practice.

ID: 35383205
Title: Dual-gRNA approach with limited off-target effect corrects C9ORF72 repeat expansion in vivo.
Abstract: C9ORF72 GGGGCC repeat expansion is the most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia, which generates abnormal DNA and RNA structures and produces toxic proteins. Recently, efficacy of CRISPR/Cas9-mediated editing has been proven in treatment of disease. However, DNA low complexity surrounding C9ORF72 expansion increases the off-target risks. Here we provide a dual-gRNA design outside of the low complexity region which enables us to remove the repeat DNA in a 'cutting-deletion-fusion' manner with a high fusion efficiency (50%). Our dual-gRNA design limits off-target effect and does not significantly affect C9ORF72 expression. In neurons carrying patient C9ORF72 expansion, our approach removes the repeat DNA and corrects the RNA foci in vitro and in vivo. Therefore, we conclude that our proof-of-concept design correct C9ORF72 repeat expansion, which may have potential therapeutic value for the patients.

ID: 36271076
Title: CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro.
Abstract: A GGGGCC24+ hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.

ID: 36409902
Title: A blood-brain penetrant RNA-targeted small molecule triggers elimination of r(G4C2)exp in c9ALS/FTD via the nuclear RNA exosome.
Abstract: A hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, or c9ALS/FTD. The RNA transcribed from the expansion, r(G4C2)exp, causes various pathologies, including intron retention, aberrant translation that produces toxic dipeptide repeat proteins (DPRs), and sequestration of RNA-binding proteins (RBPs) in RNA foci. Here, we describe a small molecule that potently and selectively interacts with r(G4C2)exp and mitigates disease pathologies in spinal neurons differentiated from c9ALS patient-derived induced pluripotent stem cells (iPSCs) and in two c9ALS/FTD mouse models. These studies reveal a mode of action whereby a small molecule diminishes intron retention caused by the r(G4C2)exp and allows the liberated intron to be eliminated by the nuclear RNA exosome, a multi-subunit degradation complex. Our findings highlight the complexity of mechanisms available to RNA-binding small molecules to alleviate disease pathologies and establishes a pipeline for the design of brain penetrant small molecules targeting RNA with novel modes of action in vivo.

ID: 38004556
Title: Rapid and Widespread Distribution of Intranasal Small Extracellular Vesicles Derived from Mesenchymal Stem Cells throughout the Brain Potentially via the Perivascular Pathway.
Abstract: Intranasal administration is a promising strategy to enhance the delivery of the sEVsomes-based drug delivery system to the central nervous system (CNS). This study aimed to explore central distributive characteristics of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) and underlying pathways. Here, we observed that intranasal MSC-sEVs were rapidly distributed to various brain regions, especially in the subcortex distant from the olfactory bulb, and were absorbed by multiple cells residing in these regions. We captured earlier transportation of intranasal MSC-sEVs into the perivascular space and found an increase in cerebrospinal fluid influx after intranasal administration, particularly in subcortical structures of anterior brain regions where intranasal sEVs were distributed more significantly. These results suggest that the perivascular pathway may underlie the rapid and widespread central delivery kinetics of intranasal MSC-sEVs and support the potential of the intranasal route to deliver MSC-sEVs to the brain for CNS therapy.

ID: 39174972
Title: Intranasal delivery of small extracellular vesicles reduces the progress of amyotrophic lateral sclerosis and the overactivation of complement-coagulation cascade and NF-ĸB signaling in SOD1G93A mice.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motoneuron degeneration, and effective clinical treatments are lacking. In this study, we evaluated whether intranasal delivery of mesenchymal stem cell-derived small extracellular vesicles (sEVs) is a strategy for ALS therapy using SOD1G93A mice. In vivo tracing showed that intranasally-delivered sEVs entered the central nervous system and were extensively taken up by spinal neurons and some microglia. SOD1G93A mice that intranasally received sEV administration showed significant improvements in motor performances and survival time. After sEV administration, pathological changes, including spinal motoneuron death and synaptic denervation, axon demyelination, neuromuscular junction degeneration and electrophysiological defects, and mitochondrial vacuolization were remarkably alleviated. sEV administration attenuated the elevation of proinflammatory cytokines and glial responses. Proteomics and transcriptomics analysis revealed upregulation of the complement and coagulation cascade and NF-ĸB signaling pathway in SOD1G93A mouse spinal cords, which was significantly inhibited by sEV administration. The changes were further confirmed by detecting C1q and NF-ĸB expression using Western blots. In conclusion, intranasal administration of sEVs effectively delays the progression of ALS by inhibiting neuroinflammation and overactivation of the complement and coagulation cascades and NF-ĸB signaling pathway and is a potential option for ALS therapy.

ID: 39233851
Title: Enhancing peptide and PMO delivery to mouse airway epithelia by chemical conjugation with the amphiphilic peptide S10.
Abstract: Delivery of antisense oligonucleotides (ASOs) to airway epithelial cells is arduous due to the physiological barriers that protect the lungs and the endosomal entrapment phenomenon, which prevents ASOs from reaching their intracellular targets. Various delivery strategies involving peptide-, lipid-, and polymer-based carriers are being investigated, yet the challenge remains. S10 is a peptide-based delivery agent that enables the intracellular delivery of biomolecules such as GFP, CRISPR-associated nuclease ribonucleoprotein (RNP), base editor RNP, and a fluorescent peptide into lung cells after intranasal or intratracheal administrations to mice, ferrets, and rhesus monkeys. Herein, we demonstrate that covalently attaching S10 to a fluorescently labeled peptide or a functional splice-switching phosphorodiamidate morpholino oligomer improves their intracellular delivery to airway epithelia in mice after a single intranasal instillation. Data reveal a homogeneous delivery from the trachea to the distal region of the lungs, specifically into the cells lining the airway. Quantitative measurements further highlight that conjugation via a disulfide bond through a pegylated (PEG) linker was the most beneficial strategy compared with direct conjugation (without the PEG linker) or conjugation via a permanent thiol-maleimide bond. We believe that S10-based conjugation provides a great strategy to achieve intracellular delivery of peptides and ASOs with therapeutic properties in lungs.

ID: 39779704
Title: Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo.
Abstract: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G4C2 repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

ID: 39800240
Title: Intranasal delivery of extracellular vesicles: A promising new approach for treating neurological and respiratory disorders.
Abstract: Extracellular vesicles (EVs) are membrane vesicles secreted by all types of cells, including bacteria, animals, and plants. These vesicles contain proteins, nucleic acids, and lipids from their parent cells and can transfer these components between cells. EVs have attracted attention for their potential use in diagnosis and therapy due to their natural properties, such as low immunogenicity, high biocompatibility, and ability to cross the blood-brain barrier. They can also be engineered to carry therapeutic molecules. EVs can be delivered via various routes. The intranasal route is particularly advantageous for delivering them to the central nervous system, making it a promising approach for treating neurological disorders. This review delves into the promising potential of intranasally administered EVs-based therapies for various medical conditions, with a particular focus on those affecting the brain and central nervous system. Additionally, the potential use of these therapies for pulmonary conditions, cancer, and allergies is examined, offering a hopeful outlook for the future of medical treatments. The intranasal administration of EVs offers significant advantages over other delivery methods. By directly delivering EVs to the brain, specifically targeting areas that have been injured, this administration proves to be highly efficient and effective, providing reassurance about the progress in medical treatments. Intranasal delivery is not limited to brain-related conditions. It can also benefit other organs like the lungs and stimulate a mucosal immune response against various pathogens due to the highly vascularized nature of the nasal cavity and airways. Moreover, it has the added benefit of minimizing toxicity to non-targeted organs and allows the EVs to remain longer in the body. As a result, there is a growing emphasis on conducting clinical trials for intranasal administration of EVs, particularly in treating respiratory tract pathologies such as coronavirus disease.

ID: 39901566
Title: Graphene Quantum Dots Attenuate TDP-43 Proteinopathy in Amyotrophic Lateral Sclerosis.
Abstract: Aberrant phase separation- and stress granule (SG)-mediated cytosolic aggregation of TDP-43 in motor neurons is the hallmark of amyotrophic lateral sclerosis (ALS). In this study, we found that graphene quantum dots (GQDs) potentially modulate TDP-43 aggregation during SG dynamics and phase separation. The intrinsically disordered region in the C-terminus of TDP-43 exhibited amyloid fibril formation; however, GQDs inhibited the formation of amyloid fibrils through direct intermolecular interactions with TDP-43. These effects were accompanied by attenuation of the ALS phenotype in animal models. Additionally, GQDs delayed the onset and survival of TDP-43 transgenic mouse models by enhancing motor neuron survival, reducing glial activation, and reducing the cytosolic aggregation of TDP-43 in motor neurons. In this research, we demonstrated the efficacy of GQDs on the SG-mediated aggregation of TDP-43 and the binding property of GQDs with TDP-43. Additionally, we demonstrated the clinical feasibility of GQDs using several animal models and other types of ALS caused by FUS and C9orf72. Therefore, GQDs could offer a new therapeutic approach for proteinopathy-associated ALS.

ID: 40409263
Title: Cross-species tropism of AAV.CPP.16 in the respiratory tract and its gene therapies against pulmonary fibrosis and viral infection.
Abstract: Efficient gene delivery vectors are crucial for respiratory and lung disease therapies. We report that AAV.CPP.16, an engineered adeno-associated virus (AAV) variant derived from AAV9, efficiently transduces airway and lung cells in mice and non-human primates via intranasal administration. AAV.CPP.16 outperforms AAV6 and AAV9, two wild-type AAVs with demonstrated tropism for respiratory tissues, and efficiently targets key respiratory cell types. It supports gene supplementation and editing therapies in two clinically relevant mouse models of respiratory and lung diseases. A single intranasal dose of AAV.CPP.16 expressing a dual-target, vascular endothelial growth factor (VEGF)/transforming growth factor (TGF)-β1-neutralizing protein protected lungs from idiopathic pulmonary fibrosis, while a similar application of AAV.CPP.16 carrying an "all-in-one" CRISPR-Cas13d system inhibited transcription of the SARS-CoV-2-derived RNA-dependent RNA polymerase (Rdrp) gene. Our findings highlight AAV.CPP.16 as a promising vector for respiratory and lung gene therapy.

ID: 40565135
Title: Perspectives in Amyotrophic Lateral Sclerosis: Biomarkers, Omics, and Gene Therapy Informing Disease and Treatment.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons, leading to muscle weakness, paralysis, and ultimately respiratory failure. Despite advances in understanding its genetic basis, particularly mutations in Chromosome 9 Open Reading Frame 72 (C9orf72), superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP), and Fused in Sarcoma (FUS) gene, current diagnostic methods result in delayed intervention, and available treatments offer only modest benefits. This review examines innovative approaches transforming ALS research and clinical management. We explore emerging biomarkers, including the fluid-based markers such as neurofilament light chain, exosomes, and microRNAs in biological fluids, alongside the non-fluid-based biomarkers, including neuroimaging and electrophysiological markers, for early diagnosis and patient stratification. The integration of multi-omics data reveals complex molecular mechanisms underlying ALS heterogeneity, potentially identifying novel therapeutic targets. We highlight current gene therapy strategies, including antisense oligonucleotides (ASOs), RNA interference (RNAi), and CRISPR/Cas9 gene editing systems, alongside advanced delivery methods for crossing the blood-brain barrier. By bridging molecular neuroscience with bioengineering, these technologies promise to revolutionize ALS diagnosis and treatment, advancing toward truly disease-modifying interventions for this previously intractable condition.

ID: 40650046
Title: Therapeutic Approaches for C9ORF72-Related ALS: Current Strategies and Future Horizons.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. One of its major genetic causes is C9ORF72, where mutations lead to hexanucleotide repeat expansions in the C9ORF72 gene. These expansions drive disease progression through mechanisms, including the formation of toxic RNAs and the accumulation of damaged proteins such as dipeptide repeats (DPRs). This review highlights these pathogenic mechanisms, focusing on RNA foci formation and the accumulation of toxic DPRs, which contribute to neuronal damage. It also discusses promising targeted therapies, including small molecules and biological drugs, designed to counteract these specific molecular events. Small molecules such as G-quadruplex stabilizers, proteasome and autophagy modulators, and RNase-targeting chimeras show potential in reducing RNA foci and DPR accumulation. Furthermore, targeting enzymes involved in repeat-associated non-AUG (RAN) translation and nucleocytoplasmic transport, which are crucial for disease pathogenesis, opens new therapeutic avenues. Even some anti-viral drugs show encouraging results in preclinical studies. Biological drugs, such as antisense oligonucleotides and gene-editing technologies like CRISPR-Cas, were explored for their potential to specifically target C9ORF72 mutations and modify the disease's molecular foundations. While preclinical and early clinical data show promise, challenges remain in optimizing delivery methods, ensuring long-term safety, and improving efficacy. This review concludes by emphasizing the importance of continued research and the potential for these therapies to alter the disease trajectory and improve patient outcomes.

ID: 40657195
Title: A Modular Bacteriophage T4 Nanoparticle Platform Enables Rapid Design of Dual COVID-19-Flu Mucosal Vaccines.
Abstract: A multivalent, rapidly deployable, mucosal vaccine platform is desperately needed to prevent acquisition and transmission of respiratory infections during epidemics and pandemics. No such approved platform currently exists and virtually all under investigation use infectious viruses that have safety concerns and are not amenable for multivalent engineering. Herein, a non-infectious biomaterial platform is presented, the bacteriophage T4 nanoparticle endowed with unique features for modular engineering, which is exploited to design dual COVID-Flu mucosal vaccines. By leveraging T4's natural affinity to nasal mucosa, in vivo CRISPR engineering, and in vitro SpyCatcher-SpyTag conjugation, hundreds of antigen molecules are incorporated from SARS-CoV-2 and influenza viruses into one nanoparticle. These include spike and hemagglutinin trimers and M2e peptides decorating the capsid while encapsulating matrix or nucleocapsid proteins inside, thereby achieving unprecedented antigen density and diversity, a pinnacle nanoparticle design. Intranasal administration of this adjuvant-free T4-CoV-Flu vaccine induces remarkable mucosal immunity against both respiratory pathogens, including high-titer neutralizing antibodies and secretory IgA, lung-resident CD4+/CD8+ T cells, diverse memory B cells, and complete protection against SARS-CoV-2 and influenza challenges. Coupled with its scalability in bacterial systems, thermostability, and adjuvant- and needle-free delivery, T4 presents an extraordinary platform to design potent mucosal vaccines against pandemic threats.

ID: 40846096
Title: Sulfonium lipid nanoparticles for intranasal mRNA delivery to lung epithelial and immune cells.
Abstract: Lung epithelial and immune cells play an important role in respiratory health, serving as the first line of defense. Targeting these cells presents significant therapeutic opportunities, particularly for mRNA-based medicine. However, efficient mRNA delivery to lung cells remains challenging due to mucosal barriers, enzymatic degradation, and complex tissue architecture. In this study, we developed sulfonium lipid nanoparticles (sLNPs) featuring a sulfonium head group and branched tail structure. These sLNPs efficiently delivered mRNA to lung epithelial and immune cells via intranasal instillation in mice, transfecting club cells, ciliated cells, and macrophages, which are key players in lung structure and function. Additionally, sLNPs successfully delivered CRISPR-Cas9 mRNA and sgRNA for genome editing, as well as cytokine mRNA for immune modulation in the lungs. The sLNP platform demonstrated safety in adult mice, with no significant local or systemic tissue damage observed. These findings highlight the sLNP platform's effectiveness and versatility in delivering diverse mRNA molecules, demonstrating its potential for applications ranging from gene editing to immunomodulation therapies. With further optimization, the sLNP system could pave the way for advanced mRNA-based treatments for lung diseases. STATEMENT OF SIGNIFICANCE: Almost all of the previously developed lipids for pulmonary mRNA delivery are amine-based. We designed and synthesized a group of lipids featuring the sulfonium charge-carrying group for mRNA delivery. This is the first demonstration of employing sulfonium lipid nanoparticles (sLNPs) for mRNA delivery to lung epithelial and immune cells in vivo. These sLNPs enabled efficient pulmonary delivery of diverse mRNA cargos, supporting applications such as bioluminescence imaging, gene editing, and immunomodulation. Club and ciliated cells as well as macrophages in the bronchoalveolar fluid, were successfully transfected. No sustained inflammation or toxicity was induced, highlighting the safety of these sulfonium lipid materials.

ID: 41076799
Title: Novel vector for efficient siRNA delivery to lymphoblasts and melanoma based on genipin-spermine nanocarriers protected with hybrid erythrocyte membrane coating.
Abstract: Efficient delivery of small interfering RNA (siRNA) remains a significant challenge in gene therapy because of the instability, poor cellular uptake, and immunogenicity of the carriers. In this study, we developed a hybrid delivery system combining genipin-spermine-glycine nanoparticles (G10S5) with erythrocyte membrane vesicles (EMVs) doped with DPPC and DSPE-PEG2000. G10S5 nanoparticles offer robust siRNA complexation and biocompatibility but may suffer from rapid clearance and immune detection. By camouflaging G10S5-siRNA polyplexes with hybrid EMVs, we aimed to increase their cell uptake and delivery efficiency. Physicochemical characterization via DLS, FTIR, TEM, cryo-EM, and AFM confirmed successful coating and favorable nanoscale morphology. Solvatochromic fluorescence analysis via the fluorescence of G10S5 indicated efficient coating. The optimized formulations at a phosphate-to‑nitrogen (P/N) ratio of 1:12 exhibited excellent RNase A resistance, strong siRNA binding, and storage stability. Compared with uncoated controls, in vitro assays demonstrated significantly enhanced cellular uptake of hybrid-coated G10S5-siRNA, with distinct internalization mechanisms. Gene silencing efficiency was validated by targeting tdTomato in tdTomato-expressing B16F10 cells, which showed effective knockdown with minimal cytotoxicity. Further validation was achieved in lymphoblastoid cell lines by targeting FARSA that has recently been implicated in C9orf72 mutation mechanism in lymphoblastoid lines. Our findings establish hybrid membrane-camouflaged G10S5 nanoparticles as promising siRNA delivery platforms, addressing the limitations of conventional carriers by leveraging their natural membrane properties and polymeric versatility. This strategy opens new avenues for the development of biomimetic, nonviral nucleic acid therapeutics.

ID: 41177462
Title: Nasal-to-brain siRNA delivery based on trace amine associated receptor for improving cognitive function.
Abstract: Gene-based therapies for central nervous system (CNS) disorders face substantial challenges in overcoming the blood-brain barrier (BBB) to effectively target brain tissues. The nasal-to-brain delivery route has gained increasing attention as it bypasses the BBB, facilitating faster drug delivery to the lesion site while minimizing systemic side effects. Here, we developed a nasal-to-brain delivery system to administer small interfering RNA (siRNA) for the treatment of radiation-induced brain injury (RBI). RNA sequencing revealed that the p53 signaling pathway was predominantly enriched in the hippocampus, with significant upregulation of Alox12B expression in RBI mice. To improve the delivery of siRNA targeting Alox12B, we engineered spermidine-modified ginseng-derived extracellular vesicles (S-GEVs) nanoparticles, termed S-GEVs@siRNA. These nanoparticles leveraged the targeting capabilities of spermidine for olfactory receptor-trace amine associated receptor (TAAR), enhancing siRNA delivery and therapeutic efficacy. After intranasal administration, the nanoparticles were efficiently internalized by olfactory receptor neurons (ORNs) via the olfactory nerve pathway. The nanoparticles then escaped lysosomes, releasing siRNA into the cytoplasm, leading to gene downregulation and therapeutic benefits. Our results demonstrated that the designed nanoparticles were absorbed by the ORNs labeled with the Olfactory Marker Protein (OMP) and TAAR5 and successfully entered the olfactory bulb and the brain. Treatment with these nanoparticles significantly reduced p53-mediated neuronal ferroptosis and improved synaptic function both in vitro and in vivo. In conclusion, S-GEVs@siRNA nanoparticles rapidly reached the olfactory bulb through TAAR-mediated endocytosis, entered hippocampal neurons, downregulated Alox12B expression, exerted neuroprotective effects, and alleviated RBI-induced cognitive dysfunction. The designed nasal-to-brain delivery system holds great promise for treating various CNS diseases.

ID: 41216864
Title: HIF-1 Targeting Intervention Renders Protection From Alzheimer's-Like Pathology in a Humanized Mice Model of HIV Infection.
Abstract: HIV-associated neurocognitive disorders (HAND) affect 30%-50% of individuals living with HIV on combination antiretroviral therapy, with Alzheimer 's-like pathology as a potent comorbidity of HAND. Our previous studies have implicated hypoxia-inducible factor-1 alpha (HIF-1α) as a central regulator of HIV-1 Tat-mediated amyloid production in astrocytes, which are further released via astrocyte-derived extracellular vesicles (ADEVs), inducing synaptodendritic injury and Alzheimer's-like pathology in naive mice. Based on this premise, we hypothesized that ADEVs carrying HIF-1α-targeting small interfering RNA (siRNA) would alleviate HIV-1-induced Alzheimer's-like pathology and neurodegeneration in CD34+ NSG HIV-infected humanized mice. Intranasally administered mCherry-TSG101-tagged ADEVs in mice demonstrated efficacy of brain delivery, especially to the hippocampus and cortex. In CD34+ NSG mice infected with HIV-1, intranasal delivery of HIF-1α siRNA-loaded ADEVs suppressed HIF-1α, reduced amyloid precursor protein (APP), AβmoC64, Aβ fibrils, and hyperphosphorylated tau (pTau), dampened glial activation as indicated by reduced GFAP and IBA1 expression, and partially restored synaptic proteins, which were dysregulated due to HIV-1 infection. Trends of improvement were also observed in behavioural deficits in spatial memory, anxiety-like behaviour, and sensorimotor gating induced by HIV-1. These findings position HIF-1α as a pivotal mediator of HIV-associated Alzheimer's-like pathology and neurodegeneration in the CD34+ NSG mice and underscore the promising role of ADEV-mediated HIF-1α siRNA delivery as a non-invasive therapeutic strategy for HAND.

ID: 41220417
Title: Ginger-Derived Exosome-Like Nanoparticles: The Effect of Extraction Methods on Metabolites and in vitro Anti-Lung Cancer Activity.
Abstract: In recent years, plant-derived exosome-like nanoparticles (PELNs) have attracted extensive attention. Among them, Ginger-derived exosome-like nanoparticles (GELNs) represent the most extensively studied category, demonstrating a wide spectrum of pharmacological activities. However, their specific efficacy against lung cancer remains largely unexplored and warrants further investigation. The appropriate isolation of GELNs is fundamental to all related research, yet a systematic comparison of different extraction methods is currently lacking. This study aimed to evaluate the differences among GELNs extracted by various methods and to investigate their anti-lung cancer pharmacological activities. The study employed four common isolation methods-ultracentrifugation (UC), sucrose gradient UC (sgUC), membrane filtration, and polyethylene glycol-based precipitation (PEG-based precipitation) - to isolate GELNs. The GELNs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and zeta potential measurements. Stability was evaluated under various conditions, including saline, serum, and different storage temperatures. The compositional profiles of GELNs extracted by four methods were explored using non-targeted metabolomics. A549 cells and PC-9 cells were used to assess the cellular uptake and anti-lung cancer efficacy of the four GELNs types. Network pharmacology, molecular docking, and molecular dynamics simulations were integrated to elucidate the potential mechanisms underlying their anti-lung cancer effects. The four methods successfully isolated GELNs with distinct profiles: UC achieved the highest protein yield (1.630 ± 0.022 g/kg), membrane filtration yielded the highest particle concentration (46.9 ± 6.71×108 particles/mL) but the lowest protein yield (0.059 ± 0.002 g/kg). Stability studies indicated that the highest stability of GELNs was observed for those isolated by UC and sgUC in both 0.9% and 10% NaCl. Furthermore, GELNs prepared by UC and membrane filtration showed excellent stability in serum. It was also demonstrated that -80°C provided the optimal storage condition for GELNs. Non-targeted metabolomics revealed the presence of 649 shared metabolites among the GELNs extracted by the four methods, along with method-specific unique metabolites. GELNs extracted by all four methods were internalized by both A549 and PC-9 cells. Among them, UC-isolated GELNs demonstrated the most potent anti-proliferative activity against the lung cancer cells. Through network pharmacology, 21 key targets of UC-isolated GELNs against lung cancer were identified. Molecular docking and molecular dynamics simulations further verified that 10-Gingerol, Hexahydrocurcumin, and [6]-Dehydrogingerdione from GELNs could stably bind to key targets, including Glycogen Synthase Kinase-3β (GSK3B), Progesterone Receptor (PGR), and SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase (SRC). This study demonstrates that although all four methods can isolate GELNs, UC is recommended for fundamental research due to its high protein yield, excellent stability, and potent in vitro anti-lung cancer activity. Furthermore, the anti-lung cancer activity of GELNs may be attributed to the regulation of GSK3B, PGR, and SRC by 10-Gingerol, Hexahydrocurcumin, and [6]-Dehydrogingerdione.

ID: 41252430
Title: Nose-to-Brain Delivery of Chlorella vulgaris Extracellular Vesicles for Antidepressant Effects.
Abstract: Current antidepressants face limitations due to the blood-brain barrier (BBB), systemic side effects and delayed onset. Here, we engineered an intranasal thermosensitive hydrogel (EVs@IN) encapsulating Chlorella vulgaris-derived extracellular vesicles (EVs) for sustained nose-to-brain delivery. EVs@IN significantly enhanced nasal mucosal retention and facilitated targeted transport of EVs to the hippocampus via olfactory pathways, while minimizing pulmonary exposure and clearance. In mouse models of depression (LPS-induced and CUMS), intranasal EVs@IN elicited rapid and potent alleviation of depressive- and anxiety-like behaviours. Mechanistically, EVs modulated astrocyte phenotypic transformation, reducing the release of neurotoxic complement C3 and suppressing neuroinflammation. Concurrently, they activated the Nrf2-Pgc-1α pathway, enhanced antioxidant defences (elevated SOD and GSH), mitigated oxidative stress and restored synaptic plasticity and neurogenesis in the hippocampus. Furthermore, we demonstrated the capacity of EVs to serve as efficient drug carriers for brain delivery. EVs@IN exhibited excellent long-term biocompatibility in vivo. Our findings establish plant-derived EVs within a sustained-release intranasal platform as a promising, scalable and BBB-bypassing strategy for the rapid treatment of depression and potentially other neuropsychiatric disorders.

ID: 41276866
Title: Cutting-edge treatments in amyotrophic lateral sclerosis: the role of molecular pathogenesis in targeted therapies.
Abstract: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the selective loss of motor neurons (MNs), leading to progressive muscle weakness, atrophy, and ultimately paralysis. This review provides a comprehensive overview of the molecular mechanisms underlying ALS pathogenesis, the genetic mutations associated with both familial and sporadic forms of the disease, and the latest therapeutic strategies aimed at mitigating disease progression. mutations in genes such as C9orf72, SOD1, TARDBP, and FUS have been implicated in ALS, with an intricate interplay of protein misfolding, oxidative stress, mitochondrial dysfunction, excitotoxicity, and neuroinflammation contributing to motor neuron degeneration. While current FDA-approved treatments such as Riluzole and Edaravone offer only modest benefits and do not significantly halt disease progression. Emerging therapies, including gene therapies (e.g., antisense oligonucleotides (ASOs) and CRISPR/Cas9, stem cell-based approaches, and neurotrophic factor supplementation, are demonstrating promising results in preclinical and early-phase clinical trials. novel approaches aim to target, modulate, and promote regeneration, renewed hope for future ALS treatments. However, several challenges remain, including effective delivery methods, safety concerns, and the inherent complexity of ALS pathology, ongoing research continues to explore these innovative interventions with the goal of improving clinical outcomes for patients. This review highlights the importance of personalized therapeutic approaches and underscores the necessity of continued innovation in ALS research, with the ultimate goal of developing disease-modifying therapies and, potentially, a cure for this fatal condition.

ID: 41277808
Title: Natural Ginger-Derived Exosomes as Effective Therapeutics for Glioblastoma.
Abstract: Despite significant therapeutic advances with chemotherapy and immunotherapy in some solid tumors, clinical outcomes for glioblastoma multiform (GBM) remain suboptimal. Owing to their high yield, easy accessibility and cost-effectiveness, plant-derived extracellular vehicles (EVs) have become attractive platforms for biomedical uses. Our study shows that fully natural ginger-derived exosomes (GEXO) effectively inhibited GBM progression through dual mechanisms: (a) direct activation of apoptotic pathways in GBM cells, and (b) induction of immunogenic cell death (ICD) that transforms dead tumor cells into endogenous vaccines. Mechanistically, GEXO demonstrated superior blood-brain barrier (BBB) penetration through clathrin-, caveolin- and macropinocytosis-mediated transcytosis, followed by tumor-specific accumulation via exocytosis. Transcriptomic analysis revealed that GEXO promoted an immunogenic shift in dying GBM cells, enhancing dendritic cell maturation and cytotoxic T-cell responses. In orthotopic GL261 and CT2A models, GEXO significantly prolonged survival without observable toxicity. The natural GEXO platform represents a promising, biosafe strategy with clinical potential for refractory GBM.

ID: 41304786
Title: Nanoparticle-Mediated Nose-to-Brain Delivery for Ischemic Stroke Therapy: Preclinical Insights.
Abstract: Ischemic stroke remains a major cause of mortality and long-term disability, yet current therapeutic strategies are largely limited to reperfusion approaches such as intravenous thrombolysis and thrombectomy, which are constrained by narrow treatment windows and the risk of complications. Moreover, the blood-brain barrier (BBB) severely restricts drug penetration into the injured brain, limiting the translation of promising neuroprotective agents into clinical success. Intranasal (IN) delivery has emerged as a compelling alternative route that bypasses the BBB and enables rapid access to the central nervous system through olfactory, trigeminal, and perivascular pathways. This narrative review highlights recent advances in preclinical research on IN therapeutics for ischemic stroke, ranging from small molecules and biologics to nucleic acids and cell-based therapies. Particular emphasis is placed on the application of nanotechnology, including extracellular vesicles, liposomes, and inorganic nanoparticles, which enhance drug stability, targeting, and bioavailability. Studies demonstrate that IN delivery of growth factors, cytokines, and engineered stem cells can promote neurogenesis, angiogenesis, white matter repair, and functional recovery, while nanocarriers further expand the therapeutic potential. Overall, intranasal delivery represents a promising and non-invasive strategy to overcome the limitations of conventional stroke therapies, offering new avenues for neuroprotection and regeneration that warrant further investigation toward clinical translation.

ID: 41310241
Title: Advances in Intranasal Delivery of Exosomes for Central Nervous System Disorders.
Abstract: Central nervous system disorders are major global health challenges that contribute to significant morbidity and mortality. Traditional therapeutic strategies often face substantial limitations, primarily due to the blood-brain barrier, which restricts the delivery of pharmacological agents to the brain and consequently affects treatment effectiveness. In recent years, in order to enhance the efficacy of the central nervous system treatments, exosome-based approaches have gained interest. Exosomes, small extracellular vesicles (30-150 nm) secreted by cells, present a feasible therapeutic strategy due to their ability to cross the blood-brain barrier and transport bioactive molecules. Reflecting the traits of their parent cells (e.g., glioma stem cells and glioblastoma multiforme), exosomes can be isolated from body fluids, which enhances their clinical applicability. Additionally, intranasal delivery provides a non-invasive method to administer exosomes, using the olfactory and trigeminal nerve pathways to bypass the blood-brain barrier and directly target the brain. This method shows great promise in enhancing therapeutic efficacy for CNS disorders. However, challenges such as rapid mucociliary clearance, enzymatic degradation, and limited bioavailability reduce efficacy. Advances in exosome engineering, nanocarrier systems, and novel delivery devices are under investigation to mitigate these constraints. However, clinical translation requires further research to guarantee safety, consistency, and scalability. In this context, intranasal exosome delivery holds considerable promise as a non-invasive strategy for central nervous system disorder treatment, contingent on overcoming the current biological and technical barriers.

ID: 41399181
Title: Engineering exosomes for Alzheimer's disease: Multi-target therapeutic strategies from pathogenesis to clinical translation.
Abstract: The complex pathogenesis of Alzheimer's disease (AD), combined with the presence of the blood‒brain barrier (BBB), severely limits the effectiveness of conventional therapeutic approaches. Engineered exosomes-nanoscale extracellular vesicles of natural origin-have emerged as a promising platform for innovative AD therapy due to their excellent biocompatibility, low immunogenicity and intrinsic ability to cross the BBB. This review provides a systematic overview of the synthetic and structural biological characteristics of exosomes, with a focus on their functionalisation through physical, chemical and genetic modifications. These approaches enable the targeted loading of therapeutic cargo and the conjugation of brain-targeting peptides, thereby facilitating precise delivery to specific brain regions and offering a multi-target therapeutic strategy for AD. We further examine the potential of engineered exosomes in modulating core AD pathological pathways, including amyloid-beta deposition, tau hyperphosphorylation, neuroinflammation and synaptic dysfunction, and highlight their utility as an integrated delivery system for the co-delivery of multiple therapeutic agents to achieve synergistic therapeutic effects. Finally, key challenges in clinical translation are addressed, such as scalable production, standardised drug loading protocols and comprehensive assessment of safety and immunogenicity. Unlike previous reviews that primarily focus on general engineering techniques, this article emphasises a rational design strategy tailored for multi-target synergistic therapy and presents a comprehensive roadmap from basic research to clinical application, thereby providing both theoretical insights and practical guidance for the development of next-generation AD treatments. KEY POINTS: A multidimensional approach combining physical, chemical, and genetic modifications equips exosomes with brain-targeted peptides, enhancing their capability for precise brain delivery in Alzheimer's disease (AD) Engineered exosomes are designed to cross the blood-brain barrier and provide stimuli-responsive release of therapeutic agents, enabling simultaneous clearance of amyloid-beta plaques and neurofibrillary tangles, and inhibition of neuroinflammation. The transition from preclinical success to early-phase human trials is underway, with intranasal administration emerging as a promising, non-invasive method for brain drug delivery. A well-defined plan for clinical translation includes scalable Good Manufacturing Practice (GMP) production, rigorous safety assessments, and biomarker-guided clinical trial design to facilitate clinical application.

ID: 41484169
Title: Plant-derived extracellular vesicles for itraconazole delivery across the blood-brain barrier for potential glioblastoma treatment.
Abstract: Background A major challenge in central nervous system disorders such glioblastoma includes the presence of a blood-brain barrier which restricts the delivery of therapeutic agents to the brain, thereby limiting the effectiveness of most conventional treatments. Moreover, the discovery of novel drugs for glioblastoma has been limited hence drug repurposing has gained traction leveraging existing drugs like itraconazole. Plant-derived extracellular vesicles (PDEVs) have potential as a natural pharmaceutical delivery system owing to their therapeutic capabilities. These PDEVs may be a good candidate for blood-brain barrier permeation due to their biomolecular composition and high drug loading efficiency of itraconazole. In this work, PDEVs isolated from aloe aborescens (aloe), Zingiber officinale (ginger) and Nigella sativa seeds [black cumin seeds (BCS)] were compared in terms of their physicochemical properties, drug release kinetics, cytotoxicity, cellular uptake in glioblastoma cells and BBB permeability. Results All PDEVs displayed nanoscale sizes ranging from 103.5 to 141 nm with negative surface charge and a spherical morphological shape observed via SEM. The drug release kinetics was assessed using different mathematical models depicting the PDEVs prolonged drug release with < 50% releasing over 21 days. The cytotoxicity studies showed that the PDEVs resulted in a higher cell viability in the non-cancerous cell line compared to A172 glioblastoma cell line. The cellular internalization of the drug showed poor uptake of blank PDEVs compared to loaded PDEVs in glioblastoma cells. The BBB permeability test showed that ginger and aloe EVs permeated the BBB whilst BCS blank and loaded EVs did not permeate the BBB. Conclusions This delivery system improves the ability of plant-derived extracellular vesicles to cross the blood-brain barrier, addressing a key challenge in delivering treatments to the brain. Through successful encapsulation of itraconazole, it paves the way for glioblastoma treatment by repurposing itraconazole with improved efficacy and reduced side effects. Furthermore, this can be incorporated in various drug delivery vehicles depending on the route of administration and therapeutic outcome i.e. intranasal, intravenous, or oral route. Future studies focus on determining the composition of PDEVs to enable engineering strategies for next generation targeting via surface modification.

ID: 41607240
Title: Engineered Biomimetic Nanorobots Orchestrate Targeted Nose-to-Brain Delivery to Resolve Neuron-Glia Entanglement against Parkinson's Disease.
Abstract: Intranasal administration enables direct brain drug delivery, showing promise for Parkinson's disease (PD) treatment. However, nose-to-brain delivery confronts sequential obstacles, including mucosal penetration, lesion-specific accumulation, and active targeting toward disease-relevant cells, demanding advanced nanotherapeutic design. Meanwhile, neural mitochondrial dysfunction and neuroinflammation constitutes two cross-interfering pathogeneses that drive PD progression. Herein, we developed an intelligent biomimetic nanoplatform (hPH‑RNPEC) based on Pueraria lobata-derived exosomes. The system is engineered with neutrophil-like membrane for inflammatory tropism, spatially staggered short unit of rabies virus glycoprotein (RVG) peptide for neuron-microglia dual targeting, and long motif of the tetrablock conjugation of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), pH-sensitive hydrazone bond, polyethylene glycol 2000 (PEG2k), and a histidine-switching peptide for efficient nasal mucosal penetration. Spatiotemporally, following intranasal administration in PD mice, hPH‑RNPEC can penetrate nasal mucosa, achieve inflammation‑directed lesion accumulation, and realize efficient cellular internalization. The system also co‑delivers endogenous exosomal miRNAs and therapeutic curcumin to mitigate neural mitochondrial damage and neuroinflammation collectively evidenced by mitochondrial function and inflammation assessment. Besides, single-cell RNA sequencing (scRNA-seq) further suggested the promotion of myelin repair and rewiring of neural circuits, which facilitate the remodeling of PD microenvironment. This study establishes an engineered biomimetic nanorobot platform for precise brain targeting and multifactorial intervention for PD treatment.

ID: 41792535
Title: Design of a Thermoresponsive Nose-to-Brain Neuromaterial for the Release of Naturally Derived Extracellular Vesicles Delivering Teriflunomide for Multiple Sclerosis.
Abstract: Multiple sclerosis is a neuroinflammatory disease characterized by demyelination and progressive neurological decline. Teriflunomide, a first-line immunomodulatory agent, faces limitations due to oral route of administration and systemic toxicity. To overcome these challenges, we developed a nose-to-brain delivery system comprising teriflunomide-loaded ginger-derived extracellular vesicles (G-EVs) embedded in an in situ nasal gel. G-EVs were isolated via serial centrifugation and double filtration and characterized for particle size (103.5 ± 1.09 nm) and zeta potential (-17.3 ± 0.32 mV) confirming nanoscale uniformity. Teriflunomide was loaded into G-EVs with an entrapment efficiency of 63.24 ± 0.75%. In vitro release studies revealed a biphasic drug release profile; an initial burst release of 3% in 24 h followed by sustained release over 21 days. The cytotoxicity of the G-EVs, loaded G-EVs and the drug was found to be non-toxic at lower concentrations (< 0.5 mg/ml). It was observed that drug loading enhanced cellular internalization of the G-EVs. Pluronic F127 and chitosan was used to formulate a thermoresponsive and mucoadhesive nasal gel. Rheological analysis demonstrated a sol-gel transition at 34.13 ± 0.76 °C, with high G' values indicating more elasticity and stiffness, behaving more like a solid. Mucoadhesion testing confirmed strong retention on mucin through texture analysis and in vitro studies. The loaded G-EVs were added to the nasal gel and SEM was performed to confirm uniformity. This formulation could offer a synergistic platform for brain drug delivery, combining the biocompatibility of naturally-derived EVs with the thermoresponsive nasal gel.

ID: 41903398
Title: Honeysuckle-derived vesicle-like nanoparticle and their hybrid vesicle as novel drug delivery systems for glioma therapy.
Abstract: Gliomas present a formidable challenge in oncology due to their immunosuppressive tumor microenvironment and the restricted delivery of therapeutics across the blood-brain barrier. Here, we report a novel hybrid nanoplatform (HEV) for synergistic chemo-immunotherapy, constructed by integrating honeysuckle-derived vesicle-like nanoparticles (HDVN) with paclitaxel (PTX)-loaded liposomes via PEG-mediated fusion. HDVN, extracted from Lonicera japonica Flos using sucrose gradient ultracentrifugation, measured 104 ± 2.1 nm in diameter and carried functional miRNAs, including miRNA2911, capable of modulating tumor-associated macrophage (TAM) polarization through the JNK and p38 MAPK pathway. The resulting HEV achieved an encapsulation efficiency of 86.58 ± 0.06% and were administered intranasally to exploit nasal-to-brain (N2B) delivery and enhanced permeability effects. In vitro, HEV exhibited potent cytotoxicity against C6 glioma cells (IC50: 3.93 µg/mL) and promoted M1 polarization of TAM, upregulating CD80, CD86, and MHC-II while suppressing CD206. In vivo, HEV significantly inhibited tumor growth in C6 glioma-bearing mice, extending median survival from 21 to 66 days, with reduced systemic toxicity compared to free paclitaxel. miRNA sequencing and KEGG pathway analysis confirmed the cross-kingdom immunomodulatory function of HDVN, contributing to the synergistic therapeutic effect. This study establishes HDVN and HEV as a pioneering nanoplatform for targeted chemo-immunotherapy in glioma, offering a promising strategy with potential for clinical translation.

ID: 41904011
Title: The quest to restore neuronal structure: Targeting cytoskeletal proteins in neurodegenerative diseases.
Abstract: Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease are characterized by progressive neuronal dysfunction and loss. A growing body of evidence implicates cytoskeletal disruption as a central pathological mechanism in these conditions. Cytoskeletal proteins, including microtubules, actin filaments, tau, neurofilaments, and alpha-synuclein, not only provide structural integrity but also regulate axonal transport, synaptic connectivity, and neuroplasticity. Its dysfunction will lead to impaired intracellular trafficking, protein aggregation, and neuronal degeneration. This chapter explores clearly about the specific cytoskeletal abnormalities that are evident in major neurodegenerative disorders, highlighting the biological mechanisms such as tauopathy-induced microtubule instability in Alzheimer's, actin cytoskeleton dysregulation in Parkinson's, and neurofilament aggregation in ALS. Current therapeutic strategies aimed at the stabilizing cytoskeletal components, enhancing protein clearance, and restoring transport dynamics are examined, alongside the cutting-edge approaches including the gene therapy, CRISPR/Cas9 editing, and nanotechnology-based delivery systems. Challenges such as limited blood-brain barrier penetration, off-target toxicity, and patient heterogeneity are also discussed with the focus on need for precision medicine. Additionally, we have also explored the future directions that specifically focused on the biomarker development, combination therapies, and strategies to promote neuroregeneration and structural plasticity. Targeting cytoskeletal pathways holds significant promise not only for suppressing the disease progression but also for rebuilding the structural foundation of the nervous system, potentially reversing the neurodegenerative decline.

ID: 41909467
Title: Exosome-like nanovesicles from acerola for CRISPR-Cas9 ribonucleoprotein delivery to the central nervous system.
Abstract: An aberrant six-base repeat in intron 1 of C9orf72 is the most frequent cause of solitary and familial amyotrophic lateral sclerosis and frontotemporal dementia. This mutation is a potential target for CRISPR/Cas9-based genome editing. However, the blood-brain barrier and limitations of current viral or nanoparticle-based delivery systems to neurons significantly restrict the clinical application of CRISPR-Cas9 in the brain. To address these challenges, we developed a drug delivery system using acerola-derived exosome-like nanoparticles (AELNs), which may overcome several limitations associated with human exosomes. AELNs stably form complexes with ribonucleoproteins (RNPs) comprised of Cas9 proteins and guide RNAs (gRNAs). We improved the delivery efficiency and selectivity of AELN/RNP complexes in GLP2-receptor-expressing neurons by incorporating GLP2 peptides into the AELN/RNP complexes. Intranasal administration of peptide-tagged AELN/RNP complexes in vivo confirmed the successful genome editing of C9orf72, demonstrating the potential of this system for treating neurodegenerative diseases. This study presents a potentially innovative approach for in vivo genome editing using a noninvasive delivery system.

ID: 41977439
Title: Targeting Non-Coding RNAs as a Potential Therapeutic and Delivery Strategy Against Neurodegenerative Diseases.
Abstract: Neurodegenerative diseases (NDs), including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), represent a growing global health challenge characterized by progressive neuronal loss and a lack of definitive disease-modifying treatments. This review explores the emerging potential of targeting non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and exosomal RNAs, to modulate pathogenic molecular pathways and address the underlying molecular origins of neurodegeneration. We evaluate the integration of advanced computational techniques for RNA structure prediction and gene regulatory network analysis, alongside chemical engineering strategies-such as Locked Nucleic Acids (LNAs) and phosphorothioate modifications-aimed at enhancing the stability and specificity of RNA-based molecules. Furthermore, we analyze cutting-edge delivery and editing technologies, including nanotechnology-driven solutions for precise neuronal targeting and the CRISPR/Cas13 system for direct ncRNA manipulation.The findings indicate that while challenges in delivery efficiency and long-term efficacy persist, the synergy of chemical engineering and computational modeling significantly improves the therapeutic profile of ncRNAs, with exosomal pathways offering a novel route for intercellular signaling modulation and biomarker discovery. Therapeutic interventions directed at specific clinical targets, such as miR-34a and BACE1-AS, demonstrate the capacity to influence protein aggregation and neuroinflammatory cascades. Although ncRNA-based therapies are currently in nascent stages, ongoing technological advancements in RNA editing and nanotechnology offer a transformative framework that could redefine the future of ND treatment and successfully halt disease progression rather than merely managing symptoms.

ID: 42053700
Title: Therapeutic Mechanisms of Stem Cell-Derived Exosomes for Neurological Disorders: An Overview.
Abstract: The current management of neurological disorders remains largely symptomatic. In recent years, stem cell-derived exosomes have emerged as a promising alternative therapeutic strategy. This narrative review synthesizes evidence from preclinical studies investigating the mechanisms and efficacy of exosome-based therapy for neurological conditions. The included studies encompass animal models and in vitro systems. Accumulating preclinical evidence consistently supports the therapeutic potential of stem cell-derived exosomes across several neurological disorders. In Alzheimer's disease models, stem cell-derived exosomes reduce β-amyloid plaque deposition and attenuate neuroinflammation. For Parkinson's disease, they exert neuroprotective effects on dopaminergic neurons. They also inhibit α-synuclein aggregation. In ischemic stroke and spinal cord injury, stem cell-derived exosomes promote functional recovery through multiple mechanisms. These include suppressing ferroptosis, promoting angiogenesis, and stimulating axonal regeneration. Improved delivery strategies, such as intranasal administration and hydrogel encapsulation, have further enhanced brain targeting and treatment durability. Despite these promising preclinical findings, several challenges remain. A primary issue is the lack of standardized preparation protocols. Significant uncertainties also exist regarding long-term safety. Furthermore, pathways for clinical translation are still unclear. Future research should prioritize elucidating the underlying mechanisms of exosome therapy. The refinement of targeted delivery systems is equally important. Finally, advancing rigorously designed clinical trials is crucial to facilitate the translation of these therapies into clinical practice.

ID: 42083346
Title: Exosomes as Advanced Nanocarriers: Overcoming the Blood-Brain Barrier for Targeted Therapeutic Delivery in Neurodegenerative Diseases.
Abstract: Exosomes, nanosized extracellular vesicles secreted by diverse cell types, have emerged as promising natural nanocarriers for therapeutic delivery. Their intrinsic ability to cross the Blood-Brain Barrier (BBB) positions them as valuable tools for treating neurodegenerative diseases. This review critically examines exosome biology, transport mechanisms, engineering strategies, and their clinical potential as drug-delivery platforms for the Central Nervous System (CNS). We analyzed recent experimental, translational, and clinical studies on exosomes and engineered derivatives, focusing on BBB penetration, therapeutic cargo delivery, and applications in brain disorders. Key advances and landmark preclinical studies were synthesized to provide a comprehensive perspective. Exosomes cross the BBB through receptor-mediated transcytosis, lipid raft-associated uptake, and macropinocytosis, enabling bidirectional transport between circulation and brain. Their intrinsic cargo, including proteins, nucleic acids, and lipids, can reflect disease states and serve as predictive biomarkers. Engineered exosomes further enhance delivery potential, as surface functionalization and optimized cargo loading improve brain specificity and therapeutic efficacy in preclinical models. Collectively, both native and engineered exosomes surpass many synthetic carriers in stability, targeting, and BBB penetration. Versus previous reviews, this manuscript integrates exosome composition, engineering, isolation technologies, and administration routes, while also addressing patent and clinical translation challenges. Importantly, it highlights quantitative and mechanistic insights into BBB transport, offering a distinct framework for advancing exosome-based CNS therapies. Exosomes constitute a versatile platform for BBB-crossing drug delivery. By consolidating mechanistic, preclinical, and translational evidence, this review highlights their transformative potential in neurodegenerative disease therapy while outlining limitations and future directions.

ID: 42126515
Title: Engineered Exosomes: Innovative Strategies for Precision Drug Delivery in Parkinson's Disease.
Abstract: Parkinson's disease is a progressive neurodegenerative disorder marked by dopaminergic neuron loss in the substantia nigra, pathological α-synuclein aggregation, and persistent neuroinflammation. Current therapies mainly offer symptomatic relief but do not halt or reverse disease progression, largely because of the restrictive blood-brain barrier. Exosomes, naturally occurring nanoscale vesicles, possess key attributes such as biocompatibility, low immunogenicity, and the capacity to cross the blood-brain barrier. In Parkinson's disease, exosomes have a dual role: they propagate α-syn pathology and amplify inflammatory signaling, accelerating disease progression; conversely, they can be engineered as carriers of neurotrophic factors, microRNAs, or small-molecule drugs, conferring neuroprotective and anti-inflammatory benefits. This review examines current strategies for exosome engineering, with emphasis on surface modification and optimized cargo loading. However, clinical translation remains hindered by suboptimal delivery efficiency, limited brain accumulation, potential immunogenicity, exosome heterogeneity, and regulatory barriers. Future research should prioritize high-affinity targeting ligands, multimodal delivery platforms, deeper insights into blood-brain barrier translocation, and integration with regenerative medicine approaches. These advancements are essential for standardized large-scale production and personalized therapies, ultimately advancing precision medicine in Parkinson's disease.

ID: 42147445
Title: Arrayed dual-gRNA CRISPR screening platform for C9orf72 repeat expansion excision in patient iPSCs.
Abstract: An intronic hexanucleotide repeat expansion in C9orf72 is the leading genetic cause of both frontotemporal dementia and amyotrophic lateral sclerosis (C9-FTD/ALS). We have previously demonstrated that CRISPR-Cas9 excision of the repeat expansion in patient iPSCs reverts pathological hallmarks of C9-FTD/ALS. Here, we aim to identify efficient and safe gRNAs for CRISPR-spCas9 dual-gRNA excision of the C9-repeat expansion. Utilizing novel ddPCR and single-molecule sequencing assays, we screened 120 gRNA pairs, comparing 64 bi-allelic, intronic excisions of the repeat region to 56 allele-specific excisions of the mutant allele in patient iPSCs, ranking them by efficiency. Bi-allelic excisions of the intronic repeat region were more efficient than excisions of the mutant allele. Single gRNA indel rates can nominate likely efficient gRNA pairs, but these pairs must be tested empirically. The length of the repeat expansion did not impact excision efficiency; rather, the activity of individual gRNAs drove excision efficiencies. Using whole genome sequencing and INDUCE-seq, we found only one detectable off-target of those nominated by Cas-OFFinder and CHANGE-seq across 4 of the most efficient gRNAs. This study advances the development of targeted therapies for C9-FTD/ALS and establishes a framework for dual-gRNA screening in patient iPSCs applicable to other repeat expansions.

ID: 42177528
Title: Engineered neuronal exosomes mediate α-synuclein clearance to ameliorate Parkinson's disease.
Abstract: Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. A hallmark pathological feature of PD is the abnormal aggregation of α-synuclein (αSyn) into insoluble Lewy bodies. Consequently, developing strategies to inhibit αSyn aggregation in the brain has been a major research focus for PD treatment. This study developed a therapeutic approach using engineered neuronal exosomes. These exosomes were modified to extend their blood circulation half-life to 3.8 h and enhance targeting, with a 2.15 ± 0.09% brain signal proportion (vs. 0.78 ± 0.07% for free dye). They were then loaded with a self-developed αSyn aggregation-blocking peptide (sPep) as well as the antioxidant pyrroloquinoline quinone (PQQ). We investigated the therapeutic efficacy of this system in both in vitro and in vivo models of PD. Our experiments confirmed that the screened sPep effectively targeted and blocked αSyn aggregation both in vitro and in vivo. Neuronal exosomes, isolated by ultracentrifugation and hybridization, demonstrated strong abilities to cross the blood-brain barrier. In vivo studies revealed that the treatment significantly improved motor and cognitive functions in PD model mice. The underlying neuroprotective mechanisms included reducing αSyn aggregation, enhancing antioxidant capacity, ameliorating mitochondrial dysfunction, and suppressing cell apoptosis, collectively promoting the survival of dopaminergic neurons. These findings demonstrate that the engineered exosome-mediated delivery system exerts a protective effect against PD pathology.

ID: 42183388
Title: Intranasal CRISPR- lipid nanoparticles targeting MAPK9 reduce neuroinflammation after traumatic brain injury.
Abstract: Traumatic brain injury (TBI) triggers a sustained neuroinflammatory response driven by activated microglia, which contributes to secondary injury and long-term neurological dysfunction. Therapeutic reprogramming of microglial activation from a pro-inflammatory (M1-like) to a reparative (M2-like) phenotype represents a promising strategy; however, the lack of cell-specific targeting within an injured brain has limited clinical translation. Here, we developed a targeted gene-editing nanotherapy to modulate post-traumatic innate immune responses. Lipid nanoparticles (LNPs) encapsulating CRISPR-Cas12a components were engineered to target mitogen-activated protein kinase-9 (MAPK9), a key regulator of pro-inflammatory signaling, and were conjugated with an Iba-1 antibody (Iba-1-CRISPR-LNPs) to enable selective targeting of microglia. In vitro, MAPK9 editing in primary macrophages inhibited M1 polarization and promoted an M2-like phenotype, leading to reduced production of pro-inflammatory cytokines. In a TBI mouse model, intranasal administration of Iba-1-CRISPR-LNPs achieved efficient delivery to the injured brain, with selective localization in Iba-1 + microglia. MAPK9 CRISPR targeting significantly attenuated microglial activation, reduced central and peripheral inflammatory responses, and decreased pro-inflammatory cytokine levels. Importantly, this approach demonstrated a favorable safety profile, with no detectable toxicity across major organs. Collectively, these findings establish a non-viral, intranasal CRISPR-based strategy for cell-specific modulation of neuroinflammation following TBI. Targeted genome editing of MAPK9 effectively reprograms microglial activation and attenuates acute inflammatory responses, highlighting its potential as a promising and translationally relevant therapeutic platform for TBI and related neuroinflammatory disorders.

ID: 42222371
Title: Exosome-based therapy for epilepsy: a systematic review and meta-analysis of preclinical studies.
Abstract: This study aims to quantitatively assess the efficacy of exosome therapy for epilepsy through a systematic review and meta-analysis of preclinical animal experiments. We seek to clarify its overall effects on seizure reduction, cognitive function preservation, and neuroinflammation suppression. A systematic search was conducted across four English-language and four Chinese databases to include epilepsy animal studies. Continuous outcomes were synthesized using standardized mean differences (SMD) and 95% confidence intervals (CI), with fixed or random effects models selected based on heterogeneity. A total of eight preclinical studies were included. The overall meta-analysis revealed that exosome treatment significantly reduced the duration of seizures (SMD = -2.30, 95% CI -4.24 to -0.36), decreased the frequency of spontaneous recurrent seizures (SMD = -1.38, 95% CI -2.17 to -0.58), and prolonged the seizure latency (SMD = 1.49, 95% CI 0.08-2.90). In terms of cognitive function, exosomes significantly shortened the escape latency in the Morris water maze (SMD = -1.38, 95% CI -2.17 to -0.58), increased the percentage of time spent in the target quadrant (SMD = 3.69, 95% CI 0.30-7.08), and enhanced the number of platform crossings (SMD = 1.41, 95% CI 0.60-2.21), with no significant changes in swimming speed. Neuropathological analysis indicated that exosome treatment significantly increased the number of hippocampal neurons (SMD = 4.48, 95% CI 1.46-7.49) and markedly reduced levels of glial fibrillary acidic protein (GFAP) (SMD = -3.61, 95% CI -7.08 to -0.14), ionized calcium-binding adaptor molecule 1 (IBA-1) (SMD = -10.27, 95% CI -20.29 to -0.25), tumor necrosis factor-alpha (TNF-α) (SMD = -2.95, 95% CI -4.21 to -1.69), and interleukin-1 beta (IL-1β) (SMD = -7.39, 95% CI -14.64 to -0.13). Although some outcomes exhibited heterogeneity and publication bias, the corrected primary effects remained statistically significant. The source of exosomes, administration route, and dosage may be critical variables influencing their efficacy. Exosome therapy improves seizure phenotypes and protects cognitive function in epilepsy models by suppressing neuroinflammation to promote neuronal survival, providing evidence for further mechanistic and clinical translation studies.

ID: 42275483
Title: Intranasal Delivery of Bacterial Extracellular Vesicles Enables RNA Cargo Entry Into the Brain.
Abstract: Extracellular vesicles (EVs) released by bacteria are potent mediators of host-microbe interactions. They modulate immune responses, deliver functional molecules and influence disease progression. However, whether bacterial EVs can access the brain and functionally affect host cells remains unclear. In this study, we engineered Escherichia coli-derived EVs by electroporating Cre recombinase mRNA (Ec EVCre) and assessed their transport and functional delivery following intranasal administration. Using mT/mG reporter mice, we observed EV uptake in the olfactory epithelium and recombination-driven GFP expression in a subset of neurons in the olfactory bulb, providing proof-of-concept for the functional delivery of bacterial EV-associated mRNA into the brain. Single-cell RNA sequencing and imaging analyses of the olfactory regions revealed neuronal and immune cell subsets as key EV targets. Microfluidic biochip chamber assays with cultured sensory neurons demonstrated that EVs undergo retrograde axonal transport from neurite terminals to the soma via signalling endosomes. Pharmacological inhibition significantly impaired EV uptake, supporting the involvement of endocytic pathways. In addition to neuronal entry, we discovered that phagocytic cells, including neutrophils and macrophages, can engulf EVCre in the nasal mucosa and migrate into the brain, providing an alternative immune-mediated route for vesicle delivery. Together, these findings indicate that bacterial EVs exploit both neuronal and phagocytic pathways to deliver functional RNA cargo into the brain, providing novel insights into microbial access to the central nervous system and its implications for neuroimmune interactions.

ID: 42292037
Title: Plant-Derived Exosome-Like Nanoparticles in Neurodegenerative Diseases: From Dual Bioactive-Delivery Roles to Translational Challenges.
Abstract: Neurodegenerative diseases, particularly Alzheimer's disease (AD) and related disorders, remain difficult to treat because of their multifactorial pathogenesis, limited disease-modifying therapies, and insufficient central nervous system exposure of many therapeutic agents. Plant-derived exosome-like nanoparticles (PELNs) are emerging as biogenic nanovesicles that combine intrinsic bioactivity with natural nanocarrier properties. Enriched with lipids, proteins, small RNAs, and phytochemicals, PELNs may exert neuroprotective effects while offering opportunities for gastrointestinal stability, systemic transport, and potential central nervous system delivery. This review critically summarizes the dual bioactive-delivery roles of PELNs in AD and related neurodegenerative disorders. We discuss their potential mechanisms in modulating neuroinflammation, glial cell-mediated immune responses, redox imbalance, mitochondrial dysfunction, pathological protein aggregation, neural repair, and gut-brain axis regulation. We further examine how administration routes, biodistribution patterns, cellular uptake, and blood-brain barrier (BBB) models influence the interpretation of evidence for central nervous system (CNS) targeting. In addition, recent advances in isolation, purification, characterization, cargo loading, and surface engineering strategies are reviewed in the context of improving stability, targeting capacity, and translational feasibility. Despite their promise, the clinical development of PELNs remains constrained by source-dependent heterogeneity, non-standardized isolation methods, insufficiently defined critical quality attributes, inconsistent dosing metrics, limited pharmacokinetic and biodistribution data, and unresolved long-term biosafety concerns. Establishing rigorous Chemistry, Manufacturing, and Controls (CMC) frameworks, reproducible quality-control assays, and evidence-based translational pathways will be essential for advancing PELNs from experimental bioactive vesicles to clinically relevant neurotherapeutic platforms.

ID: 42392306
Title: Global Trends and Evolving Frontiers in Intranasal Delivery for CNS Diseases (2000-2025): A Bibliometric Analysis and Systematic Review.
Abstract: Intranasal administration (nose-to-brain delivery) has emerged as a pivotal non-invasive strategy to bypass the blood-brain barrier (BBB) for treating central nervous system (CNS) disorders. However, the exponential growth of literature in this domain presents challenges in grasping the holistic research trajectory and identifying emerging hotspots. This study conducted a comprehensive bibliometric analysis of 4009 publications retrieved from the Web of Science Core Collection (WoSCC) spanning from 2000 to 2025. Tools including VOSviewer, CiteSpace, and R-bibliometrix were employed to map spatiotemporal trends, collaborative networks, and keyword evolution. The analysis reveals a robust upward trend in global research output, predominantly driven by China and the United States. Keyword clustering identified 5 major research sub-domains: Alzheimer's disease, Oxytocin (behavioral/psychiatric applications), Stroke and Neuroinflammation, Brain Tumors, and Nanoparticles. Burst detection analysis elucidates a distinct paradigm shift in scientific focus: early investigations prioritized mucosal absorption mechanisms and tolerance, the focus subsequently transitioned to specific therapeutic agents (e.g., insulin, oxytocin), and most recently, the field has been dominated by the optimization of delivery vectors, specifically lipid-based nanoparticles and exosomes. While nanotechnology has become the current technological frontier for enhancing brain targeting, a critical gap remains between promising preclinical results and clinical translation. Future research must prioritize the development of biomimetic delivery systems and highly predictive translational models to bridge the divide between bench and bedside. This review provides a strategic roadmap for researchers to navigate current trends and address the barriers hindering clinical application.

ID: 42507332
Title: Disease mechanisms and translational barriers guide nanocarrier design for nose to brain delivery in Alzheimer's disease.
Abstract: Alzheimer's disease (AD) is a progressive neurodegenerative disorder with limited disease-modifying treatment options, partly because many therapeutic agents show insufficient brain exposure and dose-limiting systemic adverse effects after conventional administration. Nose-to-brain (N2B) delivery has emerged as a non-invasive strategy to transport therapeutics to the central nervous system through the olfactory and trigeminal pathways, thereby partially bypassing the blood-brain barrier. Recent advances in nanomedicine and biomaterial engineering have further improved this approach by enhancing drug stability, nasal residence, mucosal transport, and brain-targeting efficiency. This review examines nanocarrier-enabled N2B delivery strategies for AD from a mechanism-guided perspective, highlighting how AD-related pathological processes shape the selection of therapeutic cargos and formulation designs. We discuss recent progress in the intranasal delivery of repurposed small molecules, natural products, insulin-related agents, peptides and proteins, extracellular vesicles, antibodies, and nucleic acid-based therapeutics. We further summarize major nanocarrier and formulation platforms, including lipid-based systems, polymeric nanoparticles, micelles, extracellular vesicles, in situ gels, and device-assisted delivery technologies. Particular attention is given to the design parameters that influence N2B performance, including particle size distribution/PDI, surface charge, mucus interaction, cargo protection, targeting modification, biodistribution, and deposition reproducibility. Finally, we critically evaluate the translational challenges that continue to limit clinical application, including species differences in nasal anatomy, dose-volume restrictions, device-dependent variability, limited human pharmacokinetic evidence, manufacturing complexity, long-term safety, and regulatory requirements. By integrating disease mechanisms, nanocarrier design, and translational considerations, this review provides a structured perspective for developing more rational and clinically feasible N2B nanodelivery systems for AD.

ID: 42524176
Title: Advances in Polyethyleneimine-Derived Nanoformulations.
Abstract: Formulations derived from polyethyleneimine (PEI) serve as versatile and efficient vehicles for the delivery of genes, drugs, and vaccines that are low-immunogenic and viable alternatives to viral vectors. PEI ensures efficient endosomal escape, preventing the therapeutic cargo from degradation, enhancing uptake, and facilitating effective cytoplasmic release via the proton sponge effect. By combining PEI with tailor-made delivery vehicles, such as polymeric assemblies, lipid-based systems, and inorganic nanomaterials, enhanced targeting, safety, and therapeutic efficacy can be accomplished. PEI-based systems are capable of delivering a wide range of drugs; in particular, they are suited to delivering drugs with a negative charge. A further function of PEI is to activate antigen-presenting cells and stimulate cytokine production in order to enable the delivery of vaccines. In spite of the promise of PEI-based formulations, biocompatibility remains a substantial concern. The most effective ways to increase PEI biocompatibility include optimizing charge density, molecular weight, and branching, developing targeted and responsive delivery systems, and using chemical modifications. To pave the way for future clinical applications, we discuss strategies to increase PEI safety, as well as recent advances and prospects in PEI-based delivery approaches for gene, drug, and vaccine delivery.

ID: 42524609
Title: In vivo delivery strategies for therapeutic CRISPR genome editing.
Abstract: CRISPR-based genome and epigenome editing technologies have rapidly evolved from programmable nucleases into a diverse therapeutic toolbox encompassing conventional CRISPR systems, base editing, prime editing, RNA targeting, and epigenetic modulation. While early clinical successes relied on ex vivo manipulation of patient-derived cells, recent advances in delivery chemistry and vector engineering are enabling direct in vivo editing across multiple organs. Here, we provide a comprehensive review of delivery modalities of CRISPR systems solely in vivo that underpin their therapeutic translation. We examine how anatomical, cellular, and immunological constraints shape organ-specific editing strategies in different organ systems and we highlight key preclinical and clinical milestones that define the current translational landscape. Across indications, delivery remains a critical determinant of efficacy, safety, and scalability, governing editor exposure, tissue selectivity, and risk of unintended genomic or epigenomic perturbation. This review, authored by members of the COST Action Genome Editing to treat Human Diseases (GenE-HumDi) Network, delineates the principles guiding in vivo genome and epigenome editing and outlines the remaining barriers to durable, tissue-selective, and broadly deployable CRISPR therapeutics.

ID: 42538925
Title: On-demand, reversible blood-brain barrier opening via electrical activation of piezoelectric nanoparticles for targeted brain drug delivery.
Abstract: Delivering therapeutics to the brain remains one of the most persistent challenges in medicine, because the blood-brain barrier (BBB) excludes over 98% of small-molecule drugs and virtually all biologics from the central nervous system (CNS). We developed electrical BBB modulation (eBBB), an on-demand platform combining vascular-targeting poly-L-lactic acid nanoparticles with high-definition transcranial direct current stimulation to achieve spatially and temporally controlled BBB opening. eBBB produced localized, reversible increases in BBB permeability confined to the stimulated cortex, with the opening area tunable via electrode geometry. This transient window enhanced regional delivery of a small-molecule drug, full-length immunoglobulins, and adeno-associated viral vectors, which are cargo classes otherwise completely excluded by the intact BBB. Neurovascular unit architecture was preserved with no lasting histological damage. Integrating a biodegradable nanomaterial with a clinically evaluated stimulation technology, eBBB offers a programmable, minimally invasive strategy for regional CNS drug delivery across brain malignancies and neurological disorders. Electrical activation of piezoelectric nanoparticles reversibly opens the blood-brain barrier for minimally invasive drug delivery to targeted cortical regions.

ID: 42549243
Title: Mechanisms and Strategies for Enhancing DNA Nuclear Entry in Gene Delivery.
Abstract: Nonviral gene delivery using DNA vectors is widely used in cell engineering, vaccination, and gene therapy, but delivery efficiency remains lower than those of viral vectors and mRNA-based approaches, partly due to inefficient nuclear entry, as transfected DNA must enter the nucleus for transcription. Therefore, a mechanistic understanding of nuclear entry pathways is essential for developing strategies to improve the efficiency. This review evaluated mechanistic studies of DNA nuclear entry in mammalian cells, mathematical models of intracellular DNA trafficking, quantitative analyses of DNA nuclear accumulation and transgene expression, and strategies to enhance nuclear delivery of DNA. Two mechanistically distinct pathways for DNA nuclear entry have been reported: enclosure upon nuclear envelope reformation in dividing cells, and active transport through nuclear pore complexes (NPCs). Various strategies have been developed to enhance nuclear import through these pathways; however, their effectiveness depends on multiple factors, including cell type, delivery methods, and cell cycle status. Although DNA vectors are significantly larger than the nominal inner diameter of NPCs, they may traverse NPCs through deformation and interactions with nuclear transport proteins. Quantitative studies show that DNA nuclear accumulation is time dependent and heterogeneous among individual cells within the same population. Nuclear entry plays a key role in determining efficiency of nonviral gene delivery. Advances in mechanistic studies, quantitative modeling, and imaging-based analyses have improved our understanding of intracellular DNA trafficking and nuclear accumulation. Integrating these insights with delivery strategies that enhance nuclear access while preserving the cellular machinery required for transgene expression will be critical for developing more efficient and reliable nonviral DNA delivery systems for therapeutic and biotechnological applications.

ID: 42567375
Title: Ginger-derived exosome-like nanoparticles incorporated into hydrogel matrix for enhanced oral delivery of celastrol to alleviate ulcerative colitis.
Abstract: Celastrol (Cel), a highly promising natural product isolated from traditional Chinese medicine, exhibits potent therapeutic efficacy against ulcerative colitis (UC). Nevertheless, its poor colon-targeting efficiency, insufficient capacity to penetrate the intestinal mucus layer, and low cellular internalization significantly compromise therapeutic outcomes in UC treatment. To address these critical limitations, herein we rationally designed a exosome-hydrogel hybrid system (Cel-GDNPs@Gel) by first encapsulating Cel into ginger-derived exosome-like nanoparticles (GDNPs), which were subsequently dispersed within a glycyrrhizic acid (GA) hydrogel matrix. Experimental studies confirmed that GDNPs were successfully isolated and characterized with uniform size distribution and round- or cup-shaped morphology, and Cel was successful encapsulated into GDNPs. The GA hydrogel endowed the system with excellent pH-sensitivity and robust mucoadhesive properties, thereby facilitating enhanced accumulation and prolonged retention at the colon site. Moreover, GDNPs promoted efficient mucus penetration and cellular uptake of Cel. Notably, both GDNPs and GA could exert synergistic therapeutic effects with Cel. Accordingly, in vitro and in vivo studies demonstrated that Cel-GDNPs@Gel significantly alleviated colitis symptoms, suppressed the expression of pro-inflammatory cytokines, attenuated oxidative stress, regulated macrophage polarization, promoted intestinal mucosal barrier repair, and restored intestinal homeostasis. Furthermore, this delivery system exhibited favorable biosafety with no obvious systemic toxicity. Collectively, this multifunctional Cel-GDNPs@Gel platform offers a safe and effective strategy for the oral treatment of UC.