Chulhee Choi

Excessive activation of NF‐κB is implicated in the pathogenesis of numerous inflammatory and autoimmune diseases; however, conventional NF‐κB inhibitors often cause widespread immunosuppression. In contrast, extracellular vesicles (EVs) are promising vehicles for therapeutic cargo delivery with advantages including reduced risk of replication. In this single‐centre, randomized, double‐blind, placebo‐controlled phase 1 trial, we evaluated ILB‐202, an engineered, allogeneic EV derived from HEK293 cells and loaded with a super‐repressor IκBα. A single ascending intravenous dose of ILB‐202 was administered to 18 healthy volunteers, and the short‐term safety, tolerability, and preliminary pharmacodynamic effects were assessed. ILB‐202 was well tolerated at all dose levels with no serious or dose‐limiting toxicities; only minor adverse events, including a mild decrease in NK cell counts and one case of grade 1 neutropenia, were observed. The laboratory parameters, vital signs and cytokine profiles remained stable, indicating no systemic immunogenicity. Single‐cell RNA sequencing revealed subtle, time‐dependent modulation of NF‐κB‐associated pathways, enhanced TGF‐β and visfatin signalling and reduced TNF signalling—suggesting a shift towards an anti‐inflammatory state. These findings support the safety and immunomodulatory activity of ILB‐202 and pave the way for future trials in diseases characterized by dysregulated NF‐κB activation.Trial Registration: ClinicalTrials.gov identifier: NCT05843799

The effectiveness of exosomes engineered to carry a dominantly active variant of inhibitor α of nuclear factor κB (NF-κB) (IκBα), super-repressor IκB (srIκB), that inhibits the expression of NF-κB in various animal models of inflammatory diseases has been demonstrated. In this study, we used a lipopolysaccharide (LPS)–induced chorioamnionitis model in pregnant nonhuman primates to explore the therapeutic potential and mode of action of srIκB-loaded exosomes (Exo-srIκBs). Intraamniotic injection of LPS induced infiltration of BCL2A1-positive neutrophils and CD68-positive macrophages in the extraplacental membranes, causing fetal lung injury. Conversely, administration of Exo-srIκB via intraamniotic and intravenous routes (6.9 × 10 ¹⁰ and 4 × 10 ¹¹ particle numbers, respectively) ameliorated these effects. Single-cell RNA sequencing of the decidua and bulk RNA sequencing of the choriodecidua highlighted that Exo-srIκB treatment mitigated LPS-induced inflammatory pathways, particularly in macrophages, leading to a cascade effect on neutrophils through NF-κB signaling inhibition. These findings underscore the potential of Exo-srIκB as a therapeutic strategy for chorioamnionitis.

Excessive activation of NF-κB is implicated in the pathogenesis of numerous inflammatory and autoimmune disease; however, conventional NF-κB inhibitors often cause widespread immunosuppression. In contrast, extracellular vesicles (EVs) are promising vehicles for therapeutic cargo delivery with advantages including reduced risk of replication. In this single-center, randomized, double-blind, placebo-controlled phase 1 trial, we evaluated ILB-202, an engineered, allogeneic EV derived from HEK293 cells and loaded with a super-repressor IκBα. A single ascending intravenous dose of ILB-202 was administered to 18 healthy volunteers, and the safety, tolerability, and preliminary pharmacodynamic effects were assessed. ILB-202 was well tolerated at all dose levels with no serious or dose-limiting toxicities; only minor adverse events, including a mild decrease in NK cell counts and one case of grade 1 neutropenia, were observed. The laboratory parameters, vital signs, and cytokine profiles remained stable, indicating no systemic immunogenicity. Single-cell RNA sequencing revealed subtle, time-dependent modulation of NF-κB-associated pathways, enhanced TGFβ and visfatin signaling and reduced TNF signaling—suggesting a shift toward an anti-inflammatory state. These findings support the safety and immunomodulatory activity of ILB-202 and pave the way for future trials in diseases characterized by dysregulated NF-κB activation. The trial is registered at ClinicalTrials.gov (ID number NCT05843799 ).

Background and Aim: Biliary atresia is a rare, progressive disease that affects the bile ducts in newborns. Persistent bile duct obstruction induces various pathological conditions, including jaundice, inflammation, and liver fibrosis; however, the exact pathogenesis of biliary atresia is not yet fully understood. Nuclear factor-κB (NF-κB) is widely acknowledged as a key regulator in the pathogenesis of hepatitis and liver fibrosis, and extensive research has been conducted to develop strategies to effectively inhibit its activity to mitigate liver damage. Exosome-based therapeutic platforms offer targeted NF-κB inhibition with low immunogenicity and enhanced liver-specific delivery. This study aimed to evaluate the therapeutic efficacy of Exo-SrIκB in treating cholestatic liver fibrosis using experimental animal models. Methods: Exo-SrIκB (an exosome-based therapy containing the super-repressor IκB protein) using EXPLOR technology (Exosome engineering for Protein Loading via Optically Reversible protein-protein interactions) to encapsulate the super repressor IκB (SrIκB) within exosomes. The therapeutic efficacy of Exo-SrIκB was assessed in minipig and mouse models with experimentally induced cholestatic liver disease. Results: Administration of Exo-SrIκB significantly attenuated liver fibrosis progression in both animal models by inhibiting NF-κB nuclear translocation and reducing the expression of fibrotic markers. Treated animals exhibited reduced collagen deposition, lower α-SMA levels, and improved hepatic function compared to untreated controls. Conclusion: Exo-SrIκB effectively suppressed NF-κB signaling and alleviated liver fibrosis in experimental cholestatic liver disease models, suggesting that exosome-based therapeutics may offer a targeted and biocompatible application to managing liver fibrosis and other chronic liver diseases.

Neuroinflammation, a significant contributor to various neurodegenerative diseases, is strongly associated with the aging process; however, to date, no efficacious treatments for neuroinflammation have been developed. In aged mouse brains, the number of infiltrating immune cells increases, and the key transcription factor associated with increased chemokine levels is nuclear factor kappa B (NF-κB). Exosomes are potent therapeutics or drug delivery vehicles for various materials, including proteins and regulatory genes, to target cells. In the present study, we evaluated the therapeutic efficacy of exosomes loaded with a nondegradable form of IκB (Exo-srIκB), which inhibits the nuclear translocation of NF-κB to suppress age-related neuroinflammation. Single-cell RNA sequencing revealed that these anti-inflammatory exosomes targeted macrophages and microglia, reducing the expression of inflammation-related genes. Treatment with Exo-srIκB also suppressed the interactions between macrophages/microglia and T and B cells in the aged brain. We demonstrated that Exo-srIκB successfully alleviates neuroinflammation by primarily targeting activated macrophages and partially modulating the functions of age-related interferon-responsive microglia in the brain. Thus, our findings highlight Exo-srIκB as a potential therapeutic agent for treating age-related neuroinflammation.

Hyperinflammation occurs in sepsis, especially in the early phase, and it could have both positive and negative effects on sepsis. Previously, we showed that a new concept of NF-κB inhibitor, exosome-based super-repressor IκBα (Exo-srIκB) delivery, has a beneficial effect on sepsis. Here, we further investigate the therapeutic effects of Exo-srIκB at different severities and phases of sepsis using an animal polymicrobial intra-abdominal infection model. We used a rat model of fecal slurry polymicrobial sepsis. First, we determined the survival effects of Exo-srIκB on sepsis according to the severity. We used two different severities of the animal sepsis model. The severe model had a mortality rate of over 50%. The mild/moderate model had a less than 30% mortality rate. Second, we administered the Exo-srIκB at various time points (1 h, 6 h, and 24 h after fecal slurry administration) to determine the therapeutic effect of Exo-srIκB at different phases of sepsis. Lastly, we determined the effects of the Exo-srIκB on cytokine production, arterial blood gas, electrolyte, and lactate. The survival gain was statistically significant in the severe sepsis model when Exo-srIκB was administered 6 h after sepsis. Interleukin 6 and interleukin-10 were significantly decreased in the kidney when administered with Exo-srIκB. The laboratory data showed that lactate, glucose, and potassium levels were significantly lowered in the NF-κB inhibitor group. In conclusion, Exo-srIκB exhibited a beneficial therapeutic effect when administered 6 h post fecal slurry administration in a severe sepsis model.

Extracellular vesicles, particularly exosomes, have emerged as promising drug delivery systems owing to their unique advantages, such as biocompatibility, immune tolerability, and target specificity. Various engineering strategies have been implemented to harness these innate qualities, with a focus on enhancing the pharmacokinetic and pharmacodynamic properties of exosomes via payload loading and surface engineering for active targeting. This concise review outlines the challenges in the development of exosomes as drug carriers and offers insights into strategies for their effective clinical translation. We also highlight preclinical studies that have successfully employed anti-inflammatory exosomes and suggest future directions for exosome therapeutics. These advancements underscore the potential for integrating exosome-based therapies into clinical practice, heralding promise for future medical interventions.

Canine atopic dermatitis (CAD) is a genetically predisposed inflammatory pruritic skin disease. The available treatments for CAD have several adverse effects and vary in efficacy, indicating the need for the development of improved treatments. In this study, we aimed to elucidate the therapeutic effects of allogeneic and xenogeneic exosomes on CAD. Six laboratory beagle dogs with CAD were randomly assigned to three treatment groups: control, canine exosome (cExos), or human exosome (hExos) groups. Dogs in the cExos and hExos groups were intravenously administered 1.5 mL of cExos (5 × 10¹⁰) and hExos (7.5 × 10¹¹) solutions, respectively, while those in the control group were administered 1.5 mL of normal saline three times per week for 4 weeks. Skin lesion score and transepidermal water loss decreased in cExos and hExos groups compared with those in the control group. The exosome treatments decreased the serum levels of inflammatory cytokines (interferon-γ, interleukin-2, interleukin-4, interleukin-12, interleukin-13, and interleukin-31) but increased those of anti-inflammatory cytokines (interleukin-10 and transforming growth factor-β), indicating the immunomodulatory effect of exosomes. Skin microbiome analysis revealed that the exosome treatments alleviated skin bacterial dysbiosis. These results suggest that allogeneic and xenogeneic exosome therapy may alleviate CAD in dogs.

Background: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA. Methods: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging. Results: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group. Conclusion: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

Jungwhoi Lee, Jungsul Lee, WooGwang Sim, Jae-Hoon Kim, Chulhee Choi & Jongwook Jeon. BMB Reports 2023;56:594-9. https://doi.org/10.5483/BMBRep.2023-0057