Exosomes are a class of extracellular vesicles that encompass a diverse array of bioactive molecules, including proteins, lipids, mRNA, and microRNA(miRNA). Virtually all cell types release exosomes under both physiological and pathological conditions. In addition to electrical and chemical signals, exosomes are an alternative route of signaling between cells in the brain. In the brain, they are involved in processes such as synaptic plasticity, neuronal stress response, intercellular communication, and neurogenesis. A number of studies have shown that exosomes regulate the occurrence and development of depression by participating in the regulation of hypothalamic-pituitary-adrenal axis, brain-derived neurotrophic factor, immune inflammatory response and other mechanisms, showing that they may become potential biological agents for the diagnosis and treatment of depression. In addition, exosomes have the ability to easily cross the blood-brain barrier, making them ideal drug or molecular delivery tools for the central nervous system. Engineered exosomes have good brain targeting ability, and their research in central nervous system diseases has begun to emerge. However, the molecular pathways involved in the pathogenesis of depression remain unknown, and further studies are needed to fully understand the role of exosomes in the development or improvement of depression. Therefore, in this review, we mainly focus on the diagnostic performance and therapeutic effect of exosomes in depression, and explore the advantages of exosomes as biomarkers and gene delivery vectors for depression.

This review delves into the role of exosomes in immune regulation within the context of preeclampsia (PE), a pregnancy condition marked by high blood pressure and widespread inflammation. PE hampers the invasion of trophoblasts and disrupts placental function, contributing to inflammation and maternal organ dysfunction. Exosomes are small extracellular vesicles that mediate cell-to-cell communication by transferring proteins, lipids, and nucleic acids. This review highlights their role in immune regulation during pregnancy, especially their altered behavior in PE. Normally, exosomes support communication between the mother and fetus, promoting immune tolerance. In PE, however, exosomal activity and content undergo significant changes, potentially intensifying the inflammatory state. Further investigation into the in vivo immune-modulatory actions of exosomes, especially those from preeclamptic placentas, may provide insights into the pathogenesis of PE and uncover novel therapeutic targets for treatment.

The nasal epithelial barrier is the first line of defense against the deep entry of pathogens or aeroallergens and is more critical in allergic rhinitis (AR). Restoring epithelial barrier dysfunction might be a promising strategy for AR. Recent studies reported that mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) potentially inhibit the inflammation response and promote tissue regeneration. However, their effect on nasal epithelial cells remains unknown.

This study sought to describe the therapeutic effect of MSC-sEV on AR, particularly focusing their effect on nasal epithelial cells and underlying molecular mechanisms.

We utilized an ovalbumin-induced mouse model to study AR. Both primary and immortalized human nasal epithelial cells (HNEpC) were used to further validate the therapeutic effects of MSC-sEV on epithelial cell function. Then we constructed microRNA (miR)-143 overexpressing and low-expressing HNEpC and MSC-sEV to elucidate molecular mechanisms. Transcriptome analysis was performed to identify the downstream pathways involved.

MSC-sEV successfully maintained nasal barrier integrity in AR mouse model. The MSC-sEV therapeutic effect on the nasal barrier was substantiated in HNEpC. Mechanistically, miR-143 was a candidate mediator of the above effects. Subsequently, transfecting HNEpC with miR-143 partially mimicked the restoring effect of MSC-sEV. MSC-sEV overexpressing miR-143 exerted more therapeutic effects on tight junctions and barrier integrity. Moreover, miR-143 regulated the glycogen synthase kinase-3β (GSK3B) pathway.

Our results indicated that MSC-sEV mitigated AR and restored nasal epithelial barrier dysfunction through the miR-143-GSK3B axis, which suggested that MSC-sEV have the remarkable ability to treat AR.

In the recent decade, extracellular vesicles (EVs) have been released from nearly all the kingdoms, modulating intercellular communication and maintaining the human body's homeostasis by regulating different cellular processes. Among EVs, exosomes are the emerging field in biopharmaceuticals. They have lipid bilayer ranging from 30 to 150 nm in size and encompass DNA, RNA, protein lipids, etc. Their sources are widespread, easy to acquire, and cost-effective in manufacturing. This review focuses on the detailed classification of exosomes existing in nature, knowledge and application of omics, therapeutic, diagnostic and theranostic application of exosomes. It covers diseases such as cancer, infectious diseases (viral, bacterial, fungal infections), neurodegenerative diseases, metabolic diseases, lifestyle diseases (diabetes, cardiovascular, gastric disorder (IBD)), autoimmune disorders and their biodistribution. This article unfolds the recent progress in the exosomes arena and covers all the regulatory considerations (FDA, EMA, and other nations) involved with it. Moreover, a detailed discussion about clinical trials and its manifestation with exosomes and challenges associated with their isolation procedures, reproducibility, and safety concerns.

Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) are nanoscale lipid bilayer vesicles secreted by mesenchymal stem cells. They inherit the parent cell's attributes, facilitating tissue repair and regeneration, promoting angiogenesis, and modulating the immune response, while offering advantages like reduced immunogenicity, straightforward administration, and enhanced stability for long-term storage. These characteristics elevate MSC-EVs as highly promising in cell-free therapy with notable clinical potential. It is critical to delve into their pharmacokinetics and thoroughly elucidate their intracellular and in vivo trajectories. A detailed summary and evaluation of existing tracing strategies are needed to establish standardized protocols. Here, we have summarized and anticipated the research progress of MSC-EVs in various biomedical imaging techniques, including fluorescence imaging, bioluminescence imaging, nuclear imaging (PET, SPECT), tomographic imaging (CT, MRI), and photoacoustic imaging. The challenges and prospects of MSC-EV tracing strategies, with particular emphasis on clinical translation, have been analyzed, with promising solutions proposed.

Neurodegenerative disorders include Parkinson's disease, spinal cord injury, multiple sclerosis and Alzheimer's disease, cause gradual neuronal loss, protein misfolding, and accumulation, resulting in severe cognitive and movement deficits. Despite substantial study, therapeutic interventions are hampered by the blood-brain barrier, which prevents medication distribution to the central nervous system. Traditional pharmaceutical methods, such as small compounds, peptides, and inhibitors, have shown minimal effectiveness in addressing this obstacle. Exosomes are nanoscale membrane-bound vesicles that are primarily engaged in intercellular communication. They have the inherent capacity to cross the blood-brain barrier, which allows them to be used as medication delivery vehicles for brain illness therapy. Exosomes may be derived from a variety of cells like microglia, astrocytes identified according to origin, increasing their flexibility as drug delivery vehicles. Advanced engineering approaches optimise exosomes for tailored distribution across the blood-brain barrier, paving the path for novel neurodegenerative disease treatments. This review discusses the promise of exosome-based drug delivery, focussing on their composition, biogenesis, engineering, and applications in treating central nervous system illnesses, eventually overcoming the unmet hurdles of crossing the blood-brain barrier.

The ability of small nucleic acids to modulate gene expression via a range of processes has been widely explored. Compared with conventional treatments, small nucleic acid therapeutics have the potential to achieve long-lasting or even curative effects via gene editing. As a result of recent technological advances, efficient small nucleic acid delivery for therapeutic and biomedical applications has been achieved, accelerating their clinical translation. Here, we review the increasing number of small nucleic acid therapeutic classes and the most common chemical modifications and delivery platforms. We also discuss the key advances in the design, development and therapeutic application of each delivery platform. Furthermore, this review presents comprehensive profiles of currently approved small nucleic acid drugs, including 11 antisense oligonucleotides (ASOs), 2 aptamers and 6 siRNA drugs, summarizing their modifications, disease-specific mechanisms of action and delivery strategies. Other candidates whose clinical trial status has been recorded and updated are also discussed. We also consider strategic issues such as important safety considerations, novel vectors and hurdles for translating academic breakthroughs to the clinic. Small nucleic acid therapeutics have produced favorable results in clinical trials and have the potential to address previously "undruggable" targets, suggesting that they could be useful for guiding the development of additional clinical candidates.

Oncogenic KRAS drives initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC). Here, we show that engineered exosomes with Kras G12D specific siRNA (iExoKras G12D ) reveal impressive biodistribution in pancreas with negligible toxicity in preclinical studies in mice and Rhesus macaques. Clinical testing of iExoKras G12D in the iEXPLORE (iExoKras G12D in Pancreatic Cancer) Phase I study employed a classical 3+3 dose escalation design (Phase Ia), followed by an accelerated titration design (Phase Ib) ( NCT03608631 ). Patients with advanced metastatic disease were enrolled after failure of multiple lines of therapy. iExoKras G12D therapy was well-tolerated with no reported dose-limiting toxicity with some cases of stable disease response, and maximum tolerated infusion was not reached even at the highest dose. Downregulation of KRAS G12D DNA and suppression of phopho-Erk was documented with increased intratumoral in CD8 + T cell infiltration in patient samples upon treatment. The CD8 + T cell recruitment priming by iExoKras G12D informed on potential efficacy of immune checkpoint therapy and lead to validation testing in preclinical PDAC models. Combination therapy of iExoKras G12D and anti-CTLA-4 antibodies, but not anti-PD1, revealed robust anti-tumor efficacy via FAS mediated CD8 + T cell anti-tumor activity. This first-in-human, precision medicine clinical trial offers new insights into priming of immunotherapy by oncogenic Kras inhibitor and an opportunistic combination therapy for PDAC patients.

Lack of sustained response to oncogenic Kras (Kras*) inhibition in preclinical models and patients with pancreatic ductal adenocarcinoma (PDAC) emphasizes the need to identify impactful synergistic combination therapies to achieve robust clinical benefit. Kras* targeting results in an influx of global T cell infiltrates including Tregs, effector CD8 + T cells and exhausted CD8 + T cells expressing several immune checkpoint molecules in PDAC. Here, we probe whether the T cell influx induced by diverse Kras* inhibitors open a therapeutic window to target the adaptive immune response in PDAC. We show a specific synergy of anti-CTLA4 immune checkpoint blockade with Kras* targeting primed by Kras G12D allele specific inhibitor, MRTX1133 and multi-selective pan-RAS inhibitor, RMC-6236, both currently in clinical testing phase. In contrast, attempted therapeutic combination following Kras* targeting with multiple checkpoint inhibitors, including anti-PD1, anti-Tim3, anti-Lag3, anti-Vista and anti-4-1BB agonist antibody failed due to compensatory mechanisms mediated by other checkpoints on exhausted CD8 + T cells. Anti-CTLA4 therapy in Kras* targeted PDAC transcriptionally reprograms effector T regs to a naïve phenotype, reverses CD8 + T cell exhaustion and is associated with recruitment of tertiary lymphoid structures (TLS) containing interferon (IFN)-stimulated/ activated B cells and germinal center B cells to enable immunotherapy efficacy and overcome resistance with long-term survival. Single cell ATAC sequencing analysis revealed that transcriptional reprogramming of Tregs is epigenetically regulated by downregulation of AP-1 family of transcription factors including Fos, Fos-b, Jun-b, Jun-d in the IL-35 promoter region. This study reveals an actionable vulnerability in the adaptive immune response in Kras* targeted PDAC with important clinical implications.

Exosomes, spherical lipid-bilayered particles secreted by cells, have recently emerged as a novel and highly promising drug delivery system, attracting extensive attention in the field of biomedical research. Dendritic-cell-derived exosomes (DC-Exos) possess surface protein and ligands characteristic of DC cells, such as functional MHC-I and MHC-II, CD80, CD86. These components play a crucial role in immune responses, facilitating antigen uptake, presentation, and the activation of antigen-specific CD4 and CD8 T cells. These properties make them striking and excellent drug delivery vehicles for use in various immune diseases and cancer therapy. This review summarizes and discusses the characteristics, current methods and types of drug loading of DC-Exos. Its surface modifications and application in disease treatment were also discussed, aiming to motivate the development of exosome-based theranostic nanoplatforms and nanotechnology for improved healthcare treatments.

Affecting a large proportion of the population worldwide, corneal disorders constitute a concerning health hazard associated to compromised eyesight or blindness for most severe cases. In the last decades, mesenchymal stem/stromal cells (MSCs) demonstrated promising abilities in improving symptoms associated to corneal diseases or alleviating these affections, especially through their anti-inflammatory, immunomodulatory and pro-regenerative properties. More recently, MSC therapeutic potential was shown to be mediated by the molecules they release, and particularly by their extracellular vesicles (EVs; MSC-EVs). Consequently, using MSC-EVs emerged as a pioneering strategy to mitigate the risks related to cell therapy while providing MSC therapeutic benefits. Despite the promises given by MSC- and MSC-EV-based approaches, many improvements are considered to optimize the therapeutic significance of these therapies. This review aspires to provide a comprehensive and detailed overview of current knowledge on corneal therapies involving MSCs and MSC-EVs, the strategies currently under evaluation, and the gaps remaining to be addressed for clinical implementation. From encapsulating MSCs or their EVs into biomaterials to enhance the ocular retention time to loading MSC-EVs with therapeutic drugs, a wide range of ground-breaking strategies are currently contemplated to lead to the safest and most effective treatments. Promising research initiatives also include diverse gene therapies and the targeting of specific cell types through the modification of the EV surface, paving the way for future therapeutic innovations. As one of the most important challenges, MSC-EV large-scale production strategies are extensively investigated and offer a wide array of possibilities to meet the needs of clinical applications.

Overview of the functions and applications of myokines and MyoEVs.

Extracellular vesicles (EVs) are heterogeneous vesicles released by donor cells that can be taken up by recipient cells, thus inducing cellular phenotype changes. Since their discovery decades ago, roles of EVs in modulating initiation, growth, survival and metastasis of cancer have been revealed. Recent studies from multifaceted perspectives have further detailed the contribution of EVs to cancer drug resistance; however, the role of EV uptake in conferring drug resistance seems to be overlooked. In this comprehensive review, we update the EV subtypes and approaches for determining EV uptake. The biological basis of EV uptake is systematically summarized. Moreover, we focus on the diverse uptake mechanisms by which EVs carry out the intracellular delivery of functional molecules and drug resistance signaling. Furthermore, we highlight how EV uptake confers drug resistance and identify potential strategies for targeting EV uptake to overcome drug resistance. Finally, we discuss the research gap on the role of EV uptake in promoting drug resistance. This updated knowledge provides a new avenue to overcome cancer drug resistance by targeting EV uptake.

Naturally occurring extracellular vesicles (EVs) and synthetic nanoparticles like liposomes have revolutionized precision diagnostics and medicine. EVs excel in biocompatibility and cell targeting, while liposomes offer enhanced drug loading capacity and scalability. The clinical translation of EVs is hindered by challenges including low yield and heterogeneity, whereas liposomes face rapid immune clearance and limited targeting efficiency. To bridge these gaps, biomimetic synthetic vesicles (SVs) have emerged as innovative platforms, combining the advantageous properties of EVs and liposomes. This review emphasizes critical aspects of EV biology, such as mechanisms of EV-cell interaction and source-dependent functionalities in targeting, immune modulation, and tissue regeneration, informing biomimetic SV engineering. We reviewed a broad array of biomimetic SVs, with a focus on lipid bilayered vesicles functionalized with proteins. These include cell-derived nanovesicles, protein-functionalized liposomes, and hybrid vesicles. By addressing current challenges and highlighting opportunities, this review aims to advance biomimetic SVs for transformative biomedical applications.

Extracellular vesicles (EVs) are cell derived nanovesicles which are implicated in both physiological and pathological intercellular communication, including the initiation, progression, and metastasis of cancer. The exchange of biomolecules between stromal cells and cancer cells via EVs can provide a window to monitor cancer development in real time for better diagnostic and interventional strategies. In addition, the process of secretion and internalization of EVs by stromal and cancer cells in the tumor microenvironment (TME) can be exploited for delivering therapeutics. EVs have the potential to provide a targeted, biocompatible, and efficient delivery platform for the treatment of cancer and other diseases. Natural as well as engineered EVs as nanomedicine have immense potential for disease intervention. Here, we provide an overview of current knowledge of EVs' function in cancer progression, diagnostic and therapeutic applications for EVs in the cancer setting, as well as current EV engineering strategies.

Extracellular vesicles (EVs) are generated in all cells. Systemic administration of allogenic EVs derived from epithelial and mesenchymal cells has been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cell-derived EVs can be modified to acquire the capacity to induce an immune response, we engineered 293T EVs to harbor the immunomodulatory molecules CD80, OX40L, and PD-L1. We demonstrated abundant levels of these proteins in the engineered cells and EVs. Functionally, the engineered EVs efficiently elicited positive and negative costimulation of human and murine T cells. In the setting of cancer and autoimmune hepatitis, the engineered EVs modulated T cell functions and altered disease progression. OX40L EVs also provided enhanced antitumor activity in combination with anti-CTLA-4 in melanoma-bearing mice. In addition, we added multiple immunomodulatory proteins in EVs (EVmIM), attempting to elicit an immune response in both lymphoid and myeloid compartments. The EVmIM containing CD80, 4-1BBL, CD40L, CD2, and CD32 engaged both T cells and antigen presenting cells (APCs) in melanoma tumors, demonstrating the capacity for EVmIM to elicit antitumor activity. Our work provides evidence that EVs can be engineered to induce specific immune responses with translational potential to modulate immune cell functions in pathological settings.

Cancer vaccines are promising therapeutic approaches to enhance specific T-cell immunity against most solid tumors. By stimulating anti-tumor immunity, clearing minimal residual disease, and minimizing adverse effects, these vaccines target tumor cells and are effective when combined with immune checkpoint blockade or other immunotherapies. However, the development of tumor cell-based vaccines faces quality issues due to poor immunogenicity, tumor heterogeneity, a suppressive tumor immune microenvironment, and ineffective delivery methods. In contrast, extracellular vesicles (EVs), naturally released by cells, are considered the ideal drug carriers and vaccine platforms. EVs offer highly organ-specific targeting, induce broader and more effective immune responses, and demonstrate superior tissue delivery ability. The development of EV vaccines is crucial for advancing cancer immunotherapy. Compared to cell-based vaccines, EV vaccines produced under Good Manufacturing Practices (GMP) offer advantages such as high safety, ease of preservation and transport, and a wide range of sources. This review summarizes the latest research findings on EV vaccine and potential applications in this field. It also highlights novel neoantigens for the development of EV vaccines against cancer.

Tissue injury repair is a multifaceted and dynamic process characterized by complex interactions among various immune cells, with M2 macrophages assuming a crucial role. Exosomes derived from M2-type macrophages (M2-Exos) significantly influence the injury repair process through intercellular communication mediated by enriched microRNAs (miRNAs). This review aims to elucidate the biological processes underlying exosome formation, the synthesis and function of miRNAs, and the diverse methodologies employed for exosome extraction. Furthermore, we provide a comprehensive summary of the established multifarious functions and mechanisms of M2-Exos miRNAs in tissue injury repair across different systems, while also exploring their potential applications in disease prevention, diagnosis, and clinical practice. Despite the challenges encountered, the therapeutic use of M2-Exos in clinical contexts appears promising, prompting research efforts to focus on improving the efficiency of exosome extraction and application, as well as ensuring the safety of their clinical utilization.

Adipose-derived mesenchymal stem cells (ADSCs) exhibit superior immunomodulatory properties and have broad therapeutic applications. They induce macrophage M2 polarization for anti-inflammatory responses. Exosomes derived from ADSCs (ADSC-EXOs) exhibit biological functions similar to those of ADSCs but can circumvent the limitations associated with cellular injection therapies. Potent anti-inflammatory substances contained in exosomes include the glycoprotein MFGE8, the cytokines such as prostaglandin E2, IL-6, and IGF, as well as non-coding nucleotides (miR-451a, miR-23, miR-30d-5p, let-7, lncRNA DLEU2, circRps5, Circ-Ptpn4, and mmu_ circ_0001359). The anti-inflammatory and immunomodulatory properties of these exosomes provide new perspectives for therapeutic approaches for graft inflammation, bone healing, acute lung injury, kidney stones, myocardial infarction, and diabetes-related diseases. This review summarizes the contents and functions of ADSC-EXOs, outlines their properties and the characteristics of macrophage phenotypes, and emphasizes their impact on macrophage polarization and their contribution to immune-related diseases.

Non-coding small molecule RNAs are associated with a variety of diseases, including infectious diseases. However, small RNA-related studies in onychomycosis have not been reported. The aim of the present study was to conduct an initial investigation of small RNA in onychomycosis. The present study collected a total of 33 affected nail samples from patients with onychomycosis and 18 normal nail samples from healthy people. Through RNA sequencing, 37 differentially expressed microRNAs (miRNAs or miRs), including 15 upregulated and 22 downregulated miRNAs, were identified in 3 patients with onychomycosis compared with 3 healthy controls. Moreover, three differentially expressed miRNAs were analyzed for further verification by RT-qPCR in other 30 affected nail and 15 healthy nail samples. Among the three verified miRNAs, a significant difference between the downregulated hsa-miR-1-3p and hsa-miR-361-3p was observed (P<0.05). A total of 14,511 target genes of 37 differentially expressed miRNAs were predicted by the miRanda and RNAhybrid databases, while the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis showed that these target genes were enriched in multiple signaling pathways. The present study indicated that hsa-miR-1-3p and hsa-miR-361-3p may be potential biomarkers for onychomycosis. Furthermore, the findings of the present study can be used in future research on RNA in onychomycosis.

During this era of rapid advancements in cancer immunotherapy, the application of cell-released small vesicles that activate the immune system is of considerable interest. Exosomes are cell-derived nanovesicles that show great promise for the immunological treatment of cancer because of their immunogenicity and molecular transfer capacity. Recent technological advancements have enabled the identification of functional functions that exosome cargoes perform in controlling immune responses. Exosomes are originated specifically from immune cells and tumor cells and they show unique composition patterns directly related to the immunotherapy against cancer. Exosomes can also deliver their cargo to particular cells, which can affect the phenotypic and immune-regulatory functions of those cells. Exosomes can influence the course of cancer and have therapeutic benefits by taking part in several cellular processes; as a result, they have the dual properties of activating and restraining cancer. Exosomes have tremendous potential for cancer immunotherapy; they may develop into the most powerful cancer vaccines and carriers of targeted antigens and drugs. Comprehending the potential applications of exosomes in immune therapy is significant for regulating cancer progression. This review offers an analysis of the function of exosomes in immunotherapy, specifically as carriers that function as diagnostic indicators for immunological activation and trigger an anti-cancer immune response. Moreover, it summarizes the fundamental mechanism and possible therapeutic applications of exosome-based immunotherapy for human cancer.

Bone marrow-derived mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) are widely used for therapeutic purposes in preclinical studies. However, their utility in treating diabetes-associated atherosclerosis remains largely unexplored. Here, we aimed to characterize BMSC-EV-mediated regulation of autophagy and macrophage polarization.

EVs were isolated from the supernatant of cultured BMSCs and characterized with transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. A diabetes-related atherosclerotic ApoE-/- mouse model was established through feeding with a high-fat diet (HFD) and streptozotocin (STZ). Histopathological analyses were carried out using Oil Red O, H&E, and Masson staining of the aorta. TEM and immunohistochemistry (IHC) were applied to evaluate autophagy, and immunofluorescence (IF) was used to identify macrophage polarization. RAW264.7 macrophages were induced with oxidized low-density lipoprotein (ox-LDL) and high glucose (HG), co-cultured with BMSC-EVs, and analyzed for macrophage proliferation, migration, and foam cell formation. RAW264.7 cells were transduced with autophagy marker mRFP-GFP-LC3 lentivirus and analyzed with IF and western blotting.

Diabetic mice (DA group) had larger aortic plaque areas and lower collagen content than the HFD mice. BMSC-EV treatment significantly reduced blood glucose, LDL levels, and aortic plaque areas while increasing collagen content. BMSC-EV-treated aortas contained a higher number of autophagosomes/autolysosomes, with increased expression of LC3BII correlating with decreased P62 levels and a lower proportion of M1 macrophages. In vitro, BMSC-EVs inhibited proliferation, migration, and foam cell formation in ox-LDL and HG-induced activated RAW264.7 cells. These effects were reversed by the autophagy blocker bafilomycin A1. Consistent with the in vivo findings, BMSC-EVs elevated levels of the autophagy-related protein LC3BII/I and decreased P62 in ox-LDL and HG-induced RAW264.7 cells. These cells also expressed the M1 macrophage markers CD86 and iNOS, but showed reduced expression of the M2 marker Arg-1. Further, BMSC-EVs decreased AMPKα and mTOR phosphorylation levels, which were blocked by the AMPK inhibitor compound C.

BMSC-EVs attenuate diabetes-exacerbated atherosclerosis by inhibiting vascular macrophage proliferation, migration, and foam cell formation via AMPK/mTOR signaling-regulated autophagy and macrophage polarization. BMSC-EVs thus hold promise as therapeutic agents for atherosclerosis.

Interest in prostate cancer as a research topic has gradually increased. As a result, a series of innovative treatment strategies have emerged with an in-depth understanding of the disease. Owing to their unique biological characteristics, mesenchymal stromal cell exosomes (MSC-Exos) have garnered significant attention for their potential to deliver targeted drugs and enable precise prostate cancer treatment. Herein, prostate cancer treatment with MSC-Exos drug-delivery systems is reviewed. This review provides a comprehensive introduction to the advantages of these systems, current research trends and progress, as well as an analysis of current challenges and future research directions. Moreover, this review lays a solid foundation for the continued development and application of MSC-Exos.

The DNA damage response (DDR) is a crucial regulatory mechanism for the survival of organisms, and irregularity of DDR may contribute to the development of various diseases, including tumors, making it is a prominent topic in therapeutic research. Extracellular vesicles (EVs), as important mediators of intercellular communication, have been extensively studied in recent years. Notably, an increasing number of studies have revealed a strong connection between DDR and EVs. On one hand, DNA damage affects the release of EVs and their compositional content; on the other hand, EVs can dictate cell survival or death by modulating DDR in both the parental and the recipient cells. This review outlines current progress in the inter-regulatory relationship between EVs and DDR, with special emphasis on the effects of EVs derived from various sources on DDR in recipient cells. In addition, the potential applications of EVs in research and tumor therapy are discussed.

Nasopharyngeal carcinoma (NPC) is a malignancy arising from the epithelium of the nasopharynx. Given its late diagnosis, NPC raises serious considerations in Southeast Asia. In addition to resistance to conventional treatment that combines chemotherapy and radiation, NPC has high rates of metastasis and frequent recurrence. Exosomes are small membrane vesicles at the nanoscale that transport physiologically active compounds from their source cell and have a crucial function in signal transmission and intercellular message exchange. The exosomes detected in the tissues of NPC patients have recently emerged as a potential non-invasive liquid biopsy biomarker that plays a role in controlling the tumor pathophysiology. Here, we take a look back at what we know so far about the complex double-edged sword role of exosomes in NPC. Exosomes could serve as biomarkers and therapeutic agents, as well as the molecular mechanisms by which they promote cell growth, angiogenesis, metastasis, immunosuppression, radiation resistance, and chemotherapy resistance in NPC. Furthermore, we go over some of the difficulties and restrictions associated with exosome use. It is anticipated that this article would provide the reference for the apply of exosomes in clinical practice.

Pancreatic ductal adenocarcinoma (PDAC) is a malignant neoplasm that typically manifests with subtle clinical manifestations in its early stages and frequently eludes diagnosis until the advanced phases of the disease. The limited therapeutic options available for PDAC significantly contribute to its high mortality rate, highlighting the urgent need for novel biomarkers capable of effectively identifying early clinical manifestations and facilitating precise diagnosis. The pivotal role of cellular exosomes in both the pathogenesis and therapeutic interventions for PDAC has been underscored. Furthermore, researchers have acknowledged the potential of exosomes as targeted drug carriers against regulatory cells in treating PDAC. The present article aims to provide a comprehensive review encompassing recent advancements in utilizing exosomes for elucidating mechanisms underlying disease development, patterns of metastasis, diagnostic techniques and treatment strategies associated with PDAC.

Exosomes, minute vesicles originating from diverse cell types, exhibit considerable potential as carriers for drug delivery in glioma therapy. These naturally occurring nanocarriers facilitate the transfer of proteins, RNAs, and lipids between cells, offering advantages such as biocompatibility, efficient cellular absorption, and the capability to traverse the blood-brain barrier (BBB). In the realm of cancer, particularly gliomas, exosomes play pivotal roles in modulating tumor growth, regulating immunity, and combating drug resistance. Moreover, exosomes serve as valuable biomarkers for diagnosing diseases and assessing prognosis. This review aims to elucidate the therapeutic and diagnostic promise of exosomes in glioma treatment, highlighting the innovative advances in exosome engineering that enable precise drug loading and targeting. By circumventing challenges associated with current glioma treatments, exosome-mediated drug delivery strategies can enhance the efficacy of chemotherapy drugs like temozolomide and overcome drug resistance mechanisms. This review underscores the multifaceted roles of exosomes in glioma pathogenesis and therapy, underscoring their potential as natural nanocarriers for targeted therapy and heralding a new era of hope for glioma treatment.

In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens with biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance. CRISPR/Cas9 plasmids were constructed, replicating in MRAB or DRPA and expressing ribonucleic proteins, leading to irreparable chromosomal lesions; however, delivering the negatively charged plasmids with extremely large molecular weight to the pathogens at the infection site became a huge challenge. We found that the BCVs integrating the bacterial out membrane vesicles and cationic lipids efficiently delivered the plasmids in vitro/in vivo to the pathogens followed by effective internalization. The BCVs were used by intratracheal or topical hydrogel application against MRAB pulmonary infection or DRPA wound infection, and both of the two pathogens were eradicated from the lung or the wound. CRISPR/Cas9 plasmid-loaded BCVs become a promising medication for drug-resistant bacteria infections.

Liver metastases frequently occur in pancreatic and colorectal cancer. Their development is promoted by tumor-derived exosomes with the integrin αVβ5 on their membrane. This integrin directs exosomes to the liver, where they promote a TGF-β-dependent pre-metastatic niche. We proposed the development of αVβ5+ exosomes to deliver anti-TGF-β therapy to the liver. This study demonstrates that the overexpression of αVβ5 in 293T cells allows its transfer to the secreted exosomes. αVβ5 overexpression increases exosome delivery to the liver, and αVβ5+ exosomes accumulate more in the liver compared to the lungs, kidneys, and brain in mice. We then sought 293T cells to directly produce and load an anti-TGF-β agent in their exosomes. First, we transduced 293T cells to express shRNAs against Tgfb1; however, the exosomes isolated from these cells did not knock down Tgfb1 in treated macrophages in vitro. However, when 293T expressed an mRNA coding a soluble form of betaglycan (sBG), a TGF-β inhibitor, this mRNA was detected in the isolated exosomes and the protein in the conditioned media of macrophages treated in vitro. In turn, this conditioned media decreased the TGF-β-induced phosphorylation of SMAD2/3 in hepatic cells in vitro. Our findings suggest that αVβ5+ exosomes could serve as nanocarriers for liver-targeted anti-TGF-β therapies.

Current approaches to oral cancer diagnosis primarily involve physical examination, tissue biopsy, and advanced computer-aided imaging techniques. However, despite these advances, patient survival rates have not significantly improved. Hence, there is a critical need to develop minimally invasive tools with high sensitivity and specificity to improve patient survival and quality of life. Liquid biopsy is a non-invasive, real-time method for predicting cancer status and potentially serves as a biomarker source for treatment response. Liquid biopsy includes rich biologically relevant components, such as circulating tumor cells, circulating tumor DNA, and extracellular vesicles (EVs). EVs are particularly intriguing due to their relatively high abundance in most biofluids, with the potential to identify specific cargo derived from circulating tumor EVs. Moreover, normal cells in lymph nodes can uptake EVs, fostering a pre-metastatic microenvironment that facilitates lymph node metastases - a common occurrence in oral cancers. This review encompasses English language publications over the last twenty years, focusing on methods for isolating EVs from saliva, blood, and lymphatic fluids, as well as the collection methods employed. Seventeen cases met the inclusion criteria according to ISEV guidelines, including 10 saliva cases, 6 blood cases, and 1 lymphatic fluid case. This review also highlighted research gaps in oral squamous cell carcinoma (OSCC) EVs, including a lack of multi-omics studies and the exploration of potential EV markers for drug resistance, as well as a notable underutilization of microfluidic technologies to translate liquid biopsy EV findings into clinical applications.

Extracellular vesicles (EVs) are heterogeneous entities secreted by cells into their microenvironment and systemic circulation. Circulating EVs carry functional small RNAs and other molecular footprints from their cell of origin, and thus have evident applications in liquid biopsy, therapeutics, and intercellular communication. Yet, the complete transcriptomic landscape of EVs is poorly characterized due to critical limitations including variable protocols used for EV-RNA extraction, quality control, cDNA library preparation, sequencing technologies, and bioinformatic analyses. Consequently, there is a gap in knowledge and the need for a standardized approach in delineating EV-RNAs. Here, we address these gaps by describing the following points by (1) focusing on the large canopy of the EVs and particles (EVPs), which includes, but not limited to - exosomes and other large and small EVs, lipoproteins, exomeres/supermeres, mitochondrial-derived vesicles, RNA binding proteins, and cell-free DNA/RNA/proteins; (2) examining the potential functional roles and biogenesis of EVPs; (3) discussing various transcriptomic methods and technologies used in uncovering the cargoes of EVPs; (4) presenting a comprehensive list of RNA subtypes reported in EVPs; (5) describing different EV-RNA databases and resources specific to EV-RNA species; (6) reviewing established bioinformatics pipelines and novel strategies for reproducible EV transcriptomics analyses; (7) emphasizing the significant need for a gold standard approach in identifying EV-RNAs across studies; (8) and finally, we highlight current challenges, discuss possible solutions, and present recommendations for robust and reproducible analyses of EVP-associated small RNAs. Overall, we seek to provide clarity on the transcriptomics landscape, sequencing technologies, and bioinformatic analyses of EVP-RNAs. Detailed portrayal of the current state of EVP transcriptomics will lead to a better understanding of how the RNA cargo of EVPs can be used in modern and targeted diagnostics and therapeutics. For the inclusion of different particles discussed in this article, we use the terms large/small EVs, non-vesicular extracellular particles (NVEPs), EPs and EVPs as defined in MISEV guidelines by the International Society of Extracellular Vesicles (ISEV).

Exosomes are vesicles produced by the human body for carrying certain information from one cell to another. The carriers are nanosized vesicles carrying a wide variety of cargo like RNA, DNA, and proteins. Exosomes are also being used in the early diagnosis of various diseases and disorders. Current research focuses on exosomes tailoring for achieving therapeutic potential in various diseases and disorders. Besides this, their biocompatibility, stability, adjustable efficacy, and targeting properties make them attractive vehicles for formulation developers. Various preclinical studies suggested that the exosome culture cells are also modified with certain genes to achieve the desirable properties of resultant exosomes. The human body also produces some other vesicles like Ectosomes and Exomeres produced along with exosomes. Additionally, vesicles like Migrasomes are produced by migrating cells and apoptotic bodies, and Oncosomes are produced by cancer cells which can also be useful for the diagnosis of various diseases and disorders. For the separation of desired exosomes from other vesicles some latest techniques that can be useful viz differential centrifugation, density gradient centrifugation, and immunoaffinity purification have been discussed. Briefly, this review summarized various techniques of isolation of purified exosomes along with an overview of the application of exosomes in various neurodegenerative disorders and cancer along with various latest aspects of exosomes in disease progression and management which might be beneficial for the researchers.

The severe respiratory effects of the coronavirus disease 2019 (COVID-19) pandemic have necessitated the immediate development of novel treatments. The majority of COVID-19-related fatalities are due to acute respiratory distress syndrome (ARDS). Consequently, this virus causes massive and aberrant inflammatory conditions, which must be promptly managed. Severe respiratory disorders, notably ARDS and acute lung injury (ALI), may be treated safely and effectively using cell-based treatments, mostly employing mesenchymal stem cells (MSCs). Since the high potential of these cells was identified, a great deal of research has been conducted on their use in regenerative medicine and complementary medicine. Multiple investigations have demonstrated that MSCs and their products, especially exosomes, inhibit inflammation. Exosomes serve a critical function in intercellular communication by transporting molecular cargo from donor cells to receiver cells. MSCs and their derived exosomes (MSCs/MSC-exosomes) may improve lung permeability, microbial and alveolar fluid clearance, and epithelial and endothelial repair, according to recent studies. This review focuses on COVID-19-related ARDS clinical studies involving MSCs/MSC-exosomes. We also investigated the utilization of Nano-delivery strategies for MSCs/MSC-exosomes and anti-inflammatory agents to enhance COVID-19 treatment.

In pancreatic ductal adenocarcinoma (PDAC), KRAS mutations drive both cancer cell growth and formation of a dense stroma. Small molecule KRAS inhibitors (KRASi) represent a breakthrough for PDAC treatment hence clinical tools that can assess early response, detect resistance and/or predict prolonged survival are desirable for management of patients undergoing KRASi therapy. We hypothesized that diffusion-weighted MRI (DWI) can detect cell death while dynamic contrast enhanced MRI (DCE) and magnetization transfer ratio (MTR) imaging are sensitive to tumor microenvironment changes, and these metrics shed insights into tumor size (standard care assessment) change induced by KRASi treatment. We tested this hypothesis in multiple preclinical PDAC models receiving MRTX1133, an investigational new drug specific for KRAS G12D mutation. Quantitative imaging markers corroborated by immunohistochemistry (IHC) revealed significant and profound changes related to cell death accompanied by changes in tumor cellularity, capillary perfusion /permeability and stromal matrix as early as 48h and day-7 after initiation of KRASi treatment, and greatly prolonged median survival over controls in a genetic engineered mouse model of PDAC (KPC). The MRI markers also captured distinct responses to KRASi therapy from PDAC tumors carrying KRAS G12C versus KRAS G12D mutation. In tumors developed resistance to MRTX1133, the imaging markers exhibited a reversal from those of responding tumors. Our findings have established that multiparametric MRI provide biological insights including cell death, reduced cellularity and tumor microenvironment changes induced by KRASi treatment and set the stage for testing the utility of these clinically ready MRI methods in patients receiving KRASi therapy.

Emerging small molecule KRAS inhibitors (KRASi) represent a new class of therapy for PDAC. Clinical tools that can provide biological insights beyond tumor size change are desirable for management of patients under KRASi therapy. DWI and DCE are frequently applied MRI methods for assessing cancer treatment responses in clinical trials. Using multiple PDAC models, we examined whether DWI, DCE and MTR can enhance the standard care assessment (tumor size) to MRTX1133, a KARSi with investigational new drug (IND) status. Our data demonstrate the abilities of DWI, DCE and MTR derived imaging markers to detect the early (48h) cell death, pronounced stromal changes and development of resistance to KRASi. This study has high translational relevance by testing clinically ready MRI methods, an IND and a genetic engineered mouse model that recapitulates saline features of human PDAC.

Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translation of EVs into viable therapeutic delivery vehicles is challenged by lengthy and inefficient processes for cargo loading and pre- and post-loading purification of EVs, resulting in limited quantity and consistency of engineered EVs.

In this work, we develop a fast and streamlined method to load surface protein-specific subpopulations of EVs with miRNA by electroporating EVs, while they are bound to antibody-coated beads. We demonstrate the selection of CD81+ EV subpopulation using magnetic microbeads, facilitating rapid EV manipulations, loading, and subsequent purification processes. Our approach shortens the time per post-electroporation EV wash by 20-fold as compared to the gold standard EV washing method, ultracentrifugation, resulting in about 2.5-h less time required to remove unloaded miRNA. In addition, we addressed the challenge of nonspecific binding of cargo molecules due to affinity-based EV selection, lowering the purity of engineered EVs, by implementing innovative strategies, including poly A carrier RNA-mediated blocking and dissociation of residual miRNA and EV-like miRNA aggregates following electroporation.

Our streamlined method integrates magnetic bead-based selection with electroporation, enabling rapid and efficient loading of miRNA into CD81+ EVs. This approach not only achieves comparable miRNA loading efficiency to conventional bulk electroporation methods but also concentrates CD81+ EVs and allows for simple electroporation parameter adjustment, promising advancements in therapeutic RNA delivery systems with enhanced specificity and reduced toxicity.

Pancreatic carcinoma is an aggressive cancer that progresses without many symptoms. The difficulty of early diagnosis and an inadequate response to traditional treatments also cause the survival rate of pancreatic cancer to be low. Current research is focusing on methods of diagnosis and treatment, such as gene therapy, to increase survival rates. Small interfering ribonucleic acid (siRNA) has emerged as a promising advanced therapeutic strategy for cancer treatment. This study sought to silence the KRAS gene in the human pancreatic carcinoma cell line using a complex of small interfering ribonucleic acid (siRNA) and gold nanoparticles (AuNP).

In this study, 25 nM siRNA and gold nanoparticles at 0.5 mg/ml, 0.25 mg/ml, and 0.125 mg/ml concentrations were used to silence the KRAS gene in the CAPAN-1 cell line. Real-time PCR analysis, agarose gel electrophoresis, and double staining were carried out, and xCelligence real-time cell analysis (RTCA) was used to measure proliferation.

The PCR analysis revealed crossing point (CP) values of actin beta (ACTB) ranging from 33.04 to 35.98, which was in the expected range for all samples. The interaction between the gold nanoparticle/siRNA complex in the double staining analysis revealed that the most effective concentration of gold nanoparticle was 0.125 mg/ml. The WST-1 technique showed that siRNA/AuPEI cells in application groups had a viability rate of over 90%, indicating no toxicity or side effects. The xCELLigence RTCA® showed that at hour 72, there was a significant difference in proliferation in the 0.5 mg/mL PEI/AuNP-siRNA, 0.25 mg/mL PEI/AuNP-siRNA, and 0.125 mg/mL PEI/AuNP-siRNA application groups compared to the control and siRNA groups (p < 0.05). By hour 96, all three groups were statistically different from the control and siRNA groups in terms of proliferation (p < 0.05).

The results of this analysis suggest that the AuPEI/siRNA complex can be effectively used to silence the target gene, but more studies are needed to verify these results.

Dry eye disease (DED), a multifactorial disease of the lacrimal system, manifests itself in patients with various symptoms such as itching, inflammation, discomfort and visual impairment. In its most severe forms, it results in the breakdown of the vital tissues of lacrimal functional unit and carries the risk of vision loss. Despite the frequency of occurrence of the disease, there are no effective curative treatment options available to date. Treatment using stem cells and its secreted factors could be a promising approach in the regeneration of damaged tissues of ocular surface. The treatment using secreted factors as well as extracellular vesicles has been demonstrated beneficial effects in various ocular surface diseases. This review provides insights on the usage of stem cell derived exosomes as a promising therapy against LG dysfunction induced ADDE for ocular surface repair.

In order to gain an overview of the existing research in this field, literature search was carried out using the PubMed, Medline, Scopus and Web of Science databases. This review is based on 164 publications until June 2024 and the literature search was carried out using the key words "exosomes", "lacrimal gland regeneration", "exosomes in lacrimal dysfunction".

The literature and studies till date suggest that exosomes and other secreted factors from stem cells have demonstrated beneficial effects on damaged ocular tissues in various ocular surface diseases. Exosomal cargo plays a crucial role in regenerating tissues by promoting homeostasis in the lacrimal system, which is often compromised in severe cases of dry eye disease. Exosome therapy shows promise as a regenerative therapy, potentially addressing the lack of effective curative treatments available for patients with dry eye disease.

Stem cell-derived exosomes represent a promising, innovative approach as a new treatment option for ADDE. By targeting lacrimal gland dysfunction and enhancing ocular surface repair, exosome therapy offers potential for significant advances in dry eye disease management. Future research is needed to refine the application of this therapy, optimize delivery methods, and fully understand its long-term efficacy in restoring ocular health.

The translation of pre-clinical anti-cancer therapies to regulatory approval has been promising, but slower than hoped. While innovative and effective treatments continue to achieve or seek approval, setbacks are often attributed to a lack of efficacy, failure to achieve clinical endpoints, and dose-limiting toxicities. Successful efforts have been characterized by the development of therapeutics designed to specifically deliver optimal and effective dosing to tumour cells while minimizing off-target toxicity. Much effort has been devoted to the rational design and application of synthetic nanoparticles to serve as targeted therapeutic delivery vehicles. Several challenges to the successful application of this modality as delivery vehicles include the induction of a protracted immune response that results in their rapid systemic clearance, manufacturing cost, lack of stability, and their biocompatibility. Extracellular vesicles (EVs) are a heterogeneous class of endogenous biologically produced lipid bilayer nanoparticles that mediate intercellular communication by carrying bioactive macromolecules capable of modifying cellular phenotypes to local and distant cells. By genetic, chemical, or metabolic methods, extracellular vesicles (EVs) can be engineered to display targeting moieties on their surface while transporting specific cargo to modulate pathological processes following uptake by target cell populations. This review will survey the types of EVs, their composition and cargoes, strategies employed to increase their targeting, uptake, and cargo release, and their potential as targeted anti-cancer therapeutic delivery vehicles.

Nucleic acid medicines are a highly attractive modality that act in a sequence-specific manner on target molecules. To date, 21 such products have been approved by the Food and Drug Administration. However, the development of nucleic acid medicines continues to face various challenges, including tissue and cell targeting as well as intracellular delivery. Numerous research groups are addressing these issues by advancing the development of nucleic acid medicines through nanotechnology. In countries other than Japan (including Europe and the USA), >40 nanotechnology-based nucleic acid medicines have been tested in clinical trials, and 15 clinical trials are ongoing. In Japan, three phase I trials are ongoing, and future results are awaited. The review summarizes the latest research in the nanotechnology of nucleic acid medicines and statuses of clinical trials in Japan, with expectations of further evolutions.

Extracellular vehicles (EVs) are gaining increasing recognition as central contributors to the intricate landscape of the tumor microenvironment (TME). This manuscript provides an extensive examination of the multifaceted roles played by EVs in shaping the TME, with a particular emphasis on their involvement in metastasis, drug resistance, and immune evasion. Metastasis, the process by which cancer cells disseminate to distant sites, remains a formidable challenge in cancer management. EVs, encompassing exosomes and microvesicles, have emerged as critical participants in this cascade of events. They facilitate the epithelial-to-mesenchymal transition (EMT), foster pre-metastatic niche establishment, and enhance the invasive potential of cancer cells. This manuscript delves into the intricate molecular mechanisms underpinning these processes, underscoring the therapeutic potential of targeting EVs to impede metastasis. Drug resistance represents a persistent impediment to successful cancer treatment. EVs are instrumental in intrinsic and acquired drug resistance, acting as mediators of intercellular communication. They ferry molecules like miRNAs and proteins, which confer resistance to conventional chemotherapy and targeted therapies. This manuscript scrutinizes the diverse strategies employed by EVs in propagating drug resistance while also considering innovative approaches involving EV-based drug delivery systems to counteract this phenomenon. Immune evasion is a hallmark of cancer, and EVs are central in sculpting the immunosuppressive milieu of the TME. Tumor-derived EVs thwart immune responses through various mechanisms, including T cell dysfunction induction, the expansion of regulatory T cells (Tregs), and polarization of macrophages towards an immunosuppressive phenotype. In addition, the manuscript explores the diagnostic potential of EVs as biomarkers and their role as therapeutic agents in immune checkpoint blockade therapies. This manuscript provides a comprehensive overview of EV's pivotal role in mediating intricate interactions within the TME, ultimately influencing cancer progression and therapeutic outcomes. A profound understanding of EV-mediated processes in metastasis, drug resistance, and immune evasion opens up promising avenues for developing innovative therapeutic strategies and identifying valuable biomarkers in the ongoing battle against cancer.

Hepatitis B virus (HBV) infection is a common cause of liver disease worldwide. The current antiviral treatment using nucleotide analogues (NAs) can only suppress de novo HBV replication but cannot eliminate chronic HBV infection due to the persistence of covalently closed circular (ccc) DNA that sustains viral replication. The CRISPR/Cas9 system is a novel genome-editing tool that enables precise gene disruption and inactivation. With high efficiency and simplicity, the CRISPR/Cas9 system has been utilized in multiple studies to disrupt the HBV genome specifically, eliciting varying anti-HBV effects both in vitro and in vivo. Additionally, multi-locus gene targeting has shown enhanced antiviral activity, paving the way for combination therapy to disrupt and inactivate HBV cccDNA as well as integrated HBV DNA. Despite its promising antiviral effects, this technology faces several challenges that need to be overcome before its clinical application, i.e., off-target effects and in vivo drug delivery. As such, there is a need for improvement in CRISPR/Cas9 efficiency, specificity, versatility, and delivery. Here, we critically review the recent literature describing the tools employed in designing guide RNAs (gRNAs) targeting HBV genomes, the vehicles used for expressing and delivering CRISPR/Cas9 components, the models used for evaluating CRISPR-mediated HBV gene disruption, the methods used for assessing antiviral and off-target effects induced by CRISPR/Cas9-mediated HBV gene disruption, and the prospects of future directions and challenges in leveraging this HBV gene-editing approach, to advance the HBV treatment toward a clinical cure.