JP7498293B2 - Use of exosome-based delivery of NF-κB inhibitors - Google Patents

Description

The present disclosure relates to a method for treating acute kidney injury using exosomes containing an NF-κB inhibitor. The present disclosure relates to a method for treating sepsis-induced disease using exosomes containing an NF-κB inhibitor.

AKI not only contributes to short-term side effects, but survivors may suffer from chronic kidney disease (CKD) and end-stage renal disease (ESRD). As one of the most important components of pathogenesis, systematic inhibition of NF-κB influences the severity of AKI. In a folic acid-induced disease model, inhibition of NF-κB reduces RelA and NF-κB2 activation, alleviating AKI damage.

Over 800 synthetic and natural materials are known to be partially involved in regulating NF-κB activation. Several studies have shown that blocking NF-κB signaling, such as blockade of the renin-angiotensin-aldosterone system (RAAS) or tumor necrosis factor-α (TNF-α) blockers, reduces kidney damage. However, these mechanisms of action are multifaceted and lack specificity. Recent advances in nanotechnology have made it possible to produce specific gene sequences or nanoparticles that target NF-κB signaling. However, no NF-κB inhibitors have yet been commercially approved for human use.

Uncontrolled inflammation is a hallmark of septic responses. Host-pathogen interactions mediated by Toll-like receptors (TLRs) stimulate the production of pro-inflammatory cytokines, chemokines, and immune activating molecules. This pro-inflammatory response is followed by a compensatory immunosuppressive response, including various quantitative and functional defects of immune cells. Pathogen-induced cellular transformation is accompanied by significant changes in host gene expression, with the nuclear factor kappa B (NF-κB) transcription factor playing a pivotal role in regulating these changes. Several cytokines under the regulatory control of NF-κB, including tumor necrosis factor (TNF)-α and interleukin (IL)-1, can induce additional activation of this transcription factor to enhance inflammatory responses and septic shock. NF-κB is also involved in apoptotic responses, mainly through enhancing the transcription of anti-apoptotic genes such as Bcl-xL, A1, and A20. Thus, inflammation associated with NF-κB activation can be exacerbated by two interacting mechanisms: increased expression of proinflammatory mediators and extended life span of cell populations, such as neutrophils, which become activated to produce proinflammatory molecules and directly participate in the acute inflammatory process.

Sepsis-induced diseases include pneumonia, cytokine storm syndrome, respiratory distress syndrome, and organ failure. Sepsis is a systemic inflammatory syndrome induced by activation of the innate immune system due to acute microbial infection and is a major cause of mortality in intensive care units. In addition, there are a variety of sepsis-induced diseases with widely different symptoms, some of which are fatal. Unfortunately, the common treatment for sepsis (i.e., antibiotics) may not be effective in treating different diseases induced by sepsis, such as cytokine syndrome or damaged organs. Currently, there is no treatment available for clinical use for sepsis-induced diseases. Therefore, there is an urgent need to develop effective alternative therapies. Although more than 700 inhibitors have been reported against NF-κB, no inhibitor has been approved as a therapeutic agent to date. Furthermore, although a variety of steroidal and nonsteroidal anti-inflammatory agents are known to block NF-κB, their effects are not specific to inhibiting NF-κB.

Successful gene and drug delivery requires the selection of an appropriate vector to stably deliver the target molecule to the site of action without adversely affecting the recipient. Various types of vectors, including recombinant adenoviruses, liposomes, ligand-conjugated nanoparticles, and ultrasonic microbubbles, have been reported to be effective for drug delivery due to their stability and high loading capacity. However, certain concerns and limitations have been associated with their use. Although they are rapidly recognized and cleared by the reticuloendothelial system, the heterogeneous particle size and non-specific absorption patterns have limited their use as biocarriers. The high immunogenicity of adenoviral vectors can induce immune responses after administration, and the virus itself can be toxic at high doses. The use of liposomes can also induce adverse immunogenicity and non-IgE-mediated hypersensitivity reactions.

Current conventional strategies for delivering therapeutic proteins include enveloping proteins in synthetic nanoparticles. The most preferred protein delivery systems are liposomes and polymeric nanoparticles (PNPs). Liposomes are synthetic vesicles with phospholipid membranes that self-assemble into various sizes and shapes in an aqueous environment. PNPs are solid colloidal particles with sizes between 10-1000 mm and are composed of biodegradable polymers to which cargo proteins can be entrapped, encapsulated or attached to the nanoparticle matrix. However, liposomes tend to fuse or aggregate with each other, resulting in premature release of liposomal cargo over time. Although PNPs may have better stability than liposomes, biocompatibility and potential long-term safety remain issues. Additionally, encapsulating proteins in liposomes and PNPs requires synthetic or recombinant protein production and an in vitro encapsulation process, whereas the EXPLOR technology requires the generation and maintenance of stable cells capable of producing cargo-loaded exosomes through natural exosome biogenesis. Exosomes also have many favorable characteristics of an ideal protein delivery system, such as biocompatibility, minimal or no inherent toxicity, long half-life in circulation, and the inherent ability to target tissues.

Recently, exosomes have received considerable attention as novel biocarriers for gene/drug delivery. Exosomes are extracellular vesicles (EVs) that play an important role in intercellular communication by delivering bioactive substances to recipient cells and influencing signaling pathways in target cells. Exosomes are easier to store and exhibit greater stability than other bioactive agents. Exosomes have a high ability to overcome biological barriers and can carry surface molecules that target specific cell types, thus inducing fewer off-target effects.

U.S. Pat. No. 1,070,2581 Korean Patent No. 10-2100420

N. Yim et al., Nature Communications 7, 12277 (2016)

The present disclosure provides a method of treating acute kidney injury (AKI) in a patient in need thereof, comprising administering to a subject an effective amount of a composition comprising an exosome that includes an NF-κB inhibitor.

The present disclosure also provides a method of treating sepsis-induced disease in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising exosomes that include an NF-κB inhibitor.

Characterization of engineered exosomes produced from HEK293T. (A) Schematic of DNA constructs used to produce super-repressor IκB-loaded exosomes (exo-srIκB) (top) and cargo protein-bearing exosome biogenesis using photoreversible protein-protein interactions, so-called EXPLOR technology (bottom). (B) Representative TEM images of exo-naive and exo-srIκB. Scale bar: 100 nm. (C) Concentration and size distribution of exo-naive (top) and exo-srIκB (bottom) were determined by Zetaview instrument. (D) Immunoblotting of exosomes from HEK293T and HEK203T cells to analyze the expression of target proteins (srIκB, mCherry, CD9 and GFP), exosome positive markers (CD63, TSG101, Alix and GAPDH) and exosome negative markers (organelle markers; prohibitin, calnexin, GM130 and nucleoporin p62). Naive and exo-naive were used as negative controls for exo-srIκB. Renal protective effect of exo-srIκB after renal IRI. (A) Experimental scheme of renal IRI surgery and exosome transfer. Each mouse group was intraperitoneally injected with 3×10 ⁹ pn of exo-naive or exo-srIκB three times at 1-h intervals (total of 9×10 ⁹ pn). Mice were sacrificed 24 or 48 h after IRI surgery, and serum and tissues were collected for further evaluation. (B-D) Serum levels of BUN, creatinine and NGAL (24 h and 48 h) among groups differing by treatment type (exo-naive vs. exo-srIκB), drug delivery time (pre-treatment vs. post-treatment) and follow-up time point, demonstrating the renal protective effect of exo-srIκB treatment (pre-treatment 24-h, n=10; pre-treatment 48-h, n=4-5; post-treatment 24-h, n=5-10; post-treatment 48-h, n=4-11). (E) Left: Representative PAS staining images of cortical tubular cells in kidney sections of each group. Normal proximal ductal brush border ( ^* ) or loss of brush border (o); chromatin condensation (black arrow); denuded basement membrane (black arrow); vacuumization (yellow arrow); Scale bar, 100 μM. Right: pathological ductal damage; multiple kidney samples from each group show less ductal damage with exo-srIκB treatment than with exo-naive treatment (pre-treatment 24-h, n=5; post-treatment 24-h, n=5). Comparisons between groups were assessed using one-way ANOVA with Bonferroni post-hoc tests. Data presented as mean ± SD values. ***P<0.001 comparing exo-naive-sham and exo-naive-IRI surgery groups. #P<0.05 and ###P<0.001 when comparing exo-naive-IRI and exo-srIκB-IRI surgery groups. Inhibition of IRI-induced NF-κB activation after exo-srIκB treatment. (A) Western blot analysis of NF-κB p65 expression using kidney nuclear extracts from each experimental mouse group. Nuclear extracts were biochemically separated from cytoplasmic fractions, and NF-κB p65 and lamin B1 expression were analyzed via Western blotting. IRI-induced activation of NF-κB signaling was significantly inhibited by exo-srIκB treatment before and after surgery. (B) Increased DNA binding activity of NF-κB p65 after renal IRI was inhibited by exo-srIκB treatment before and after surgery. (C) Left: Representative IHC images using NF-κB p65 antibody from each treatment group. Scale bar, 50 μM. Right: Graphical representation showing immunohistochemical detection of NF-κB p65 in each experimental group. Treatment with exo-srIκB reduced NF-κB expression compared to the control group (pre-treatment 24-h, n=5-8; pre-treatment 48-h, n=4-5; post-treatment 24-h, n=5-10; post-treatment 48-h, n=4-11). Comparisons between groups were assessed using one-way ANOVA with Bonferroni post-hoc test. Data are expressed as mean ± SD values. **P<0.01, ***P<0.001 for comparing exo-naive-sham and exo-naive-IRI-surgery groups. #P<0.05, ##P<0.01, ###P<0.001 for comparing exo-naive-IRI-surgery and exo-srIκB-IRI-surgery groups. Expression of proinflammatory cytokines/chemokines and adhesion molecules was adversely affected by exo-srIκB treatment both locally and systemically. (A) Total kidney lysates from each experimental group were used for qRT-PCR to measure the expression levels of proinflammatory cytokines including IL-1β, IL-6, Tnf-α, Ccl2, Ccl5, and Cxc12. The mRNA levels of such genes were significantly increased after IR injury in the kidney, and this effect was mitigated by exo-srIκB treatment (pre-treatment 24-h, n=5-8; pre-treatment 48-h, n=4-5; post-treatment 24-h, n=5-10; post-treatment 48-h, n=4-11). (B) Multiplex cytokine studies using serum from each experimental group also showed a similar trend towards decreased proinflammatory cytokine levels in postischemic mouse groups with exo-srIκB treatment. Expression of proinflammatory cytokines/chemokines and adhesion molecules is adversely affected by exo-srIκB treatment both locally and systemically. (C) qRT-PCR data show increased levels of Icam-1 mRNA in the exo-naive treated group after ischemia and a significant decrease in Icam-1 mRNA with exo-srIκB treatment. (D) Western blot analysis of total kidney lysates from each group demonstrated decreased expression of ICAM-1 in IR injured kidneys with exo-srIκB treatment. (E) Representative kidney sections showing ICAM-1 IHC staining (left) and a graphical representation of ICAM-1 IHC (right) show that exo-srIκB treatment decreased ICAM-1 expression in the kidneys after ischemia in C57BL/6J mice. Scale bar, 50 μM (B-E: pre-treatment 24-h, n=5; pre-treatment 48-h, n=4-5; post-treatment 24-h, n=5; post-treatment 48-h, n=3-6). Comparisons between groups were assessed using one-way ANOVA with Bonferroni post-hoc test. Data are expressed as mean ± SD values. *P<0.05, **P<0.01, ***P<0.001 when comparing exo-naive-sham and exo-naive IRI surgery groups. #P<0.05, ##P<0.01, ###P<0.001 when comparing exo-naive-IRI and exo-srIκB-IRI surgery groups. Exo-srIκB treatment ameliorates IR-induced renal apoptosis. (A) Left: Western blot analysis comparing cleaved caspase-3 and cleaved PARP protein expression in kidneys from each experimental group. Right: Graphical representation shows that exo-srIκB treatment reduced the levels of cleaved caspase-3 and cleaved PARP compared to exo-naive treatment. (B) Left: Representative images of TUNEL staining (FITC labeling) are presented. Apoptotic cells are green (white arrows) and DAPI was used as counterstain. Scale bar, 50 μM. Right: Bar graph showing TUNEL positive cells in kidney sections from each group. Exo-srIκB treatment induced a lower number of apoptotic cells (pre-treatment 24-h, n=5; pre-treatment 48-h, n=4-5; post-treatment 24-h, n=5; post-treatment 48-h, n-3-6). Comparisons between groups were assessed using one-way ANOVA with Bonferroni post-hoc test. Data are expressed as mean ± SD values. ***P<0.001 comparing exo-naive-sham and exo-naive-IRI surgery groups. #P<0.05, ##P<0.01 and ###P<0.001 comparing exo-naive-IRI and exo-srIκB IRI surgery groups. Biodistribution of exosomes after renal ischemia-reperfusion injury. (a) Experimental scheme of in vivo imaging and exosome delivery. (b) Biodistribution of exosomes after renal ischemia-reperfusion injury. (b) Stepwise intravital imaging shows uptake of DiD-labeled exosomes (green) into neutrophils (Ly6G ⁺ , red) and macrophages (F4/80 ⁺ , blue) in postischemic kidneys treated with venous exo-srIκB. White arrows indicate DiD-labeled exosomes contained within immune cells. White wavy lines indicate renal interstitium. (c) Biodistribution of DiD-labeled exosomes (green) in the spleen after renal IRI surgery shows uptake of exosomes into neutrophils (Ly6G ⁺ , red) and macrophages (F4/80 ⁺ blue) in the external parenchyma. Time lapse is indicated. Scale bar, 20 μm. Exo-srIκB treatment modulates renal immune cell populations following IR-induced AKI. (A) Mice were sacrificed 24 hours after surgery. KMNCs were enriched using enzymatic digestion, mechanical disruption, and Percoll density gradient. Total renal cell numbers determined using these methods showed no statistical differences between experimental groups. (B) Graphical representation of flow cytometry showed a higher proportion of renal CD45 ⁺ cells following IRI among renal cells isolated using enzymatic digestion, mechanical disruption, and Percoll density gradient. Exo-srIκB treatment attenuated the postischemic surge of renal immune cells. (C) Additional staining with additional immune cell markers showed that the frequencies of multilineage immune cells, including neutrophils (CD45 ⁺ Ly6G ⁺ ), pro-/anti-inflammatory mononuclear phagocytes (CD45 ⁺ Ly6C ⁺ /CD45 ⁺ F4/80 ⁺ ), and T cells (CD45 ⁺ CD3 ⁺ ) in enriched KMNCs, were also decreased by exo-srIκB treatment in the postischemic kidney. Exo-srIκB treatment modulates renal immune cell populations after IR-induced AKI. (D-F) Immunofluorescence studies were performed on postischemic kidneys from each experimental group targeting Ly6G, Ly6C, and F4/80. Secondary antibodies conjugated with Alexa Fluor 647 were used for all immunofluorescence experiments. Data show a decreased frequency of Alexa Fluor 647 stained cells (white arrows) in postischemic kidneys after exo-srIκB treatment, suggesting that there were fewer neutrophils (Ly6G ⁺ ) and exo-srIκB pro/anti-inflammatory mononuclear phagocytes (Ly6C ⁺ /F4/80 ⁺ ) in exo-srIκB-treated kidneys. Proximal duct cells were stained with LTL (green) and DAPI was used as counterstain. Scale bar, 50 μM, (n=5 per experimental group). Comparisons between groups were assessed using one-way ANOVA with Bonferroni post-hoc test. Data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 for comparison of exo-naive-sham and exo-naive-IRI surgery groups. #P<0.05, ##P<0.01 for comparison of exo-naive-IRI and exo-srIκB-IRI surgery groups. Intravenous delivery of exo-srIκB exerts similar biological effects compared to intraperitoneal delivery in an ischemic AKI model. (a) Experimental scheme of renal IRI surgery and exosome delivery. Each mouse group was intravenously injected with 9×10 ⁹ pn exo-naive, exo-srIκB, or PBS 1 h after reperfusion. Mice were sacrificed 24 or 48 h after IRI surgery, and serum and tissues were collected for further evaluation. (b-d) Serum levels of BUN, creatinine, and NGAL between groups differing by treatment type (PBS vs. exo-naive vs. exo-srIκB) and follow-up observation time points (24-h and 48-h), showing the renal protective effect of exo-srIκB treatment (n=5 per experimental group). (e) Western blot analysis of NF-κB p65 expression using kidney nuclear extracts from each experimental mouse group. Nuclear extracts were biochemically separated from cytoplasmic fractions, and NF-κB p65 and lamin B1 expression was analyzed via Western blotting. IRI-induced activation of NF-κB signaling was significantly suppressed by exo-srIκB treatment after surgery. (f) The elevated DNA binding activity of NF-κB p65 after renal IRI was suppressed by exo-srIκB treatment after surgery. Intravenous delivery of exo-srIκB exerts similar biological effects compared to intraperitoneal delivery in the ischemic AKI model. (g) qRT-PCR data show increased levels of Icam-1 mRNA in the exo-naive treated group after ischemia and a significant decrease in Icam-1 mRNA by exo-srIκB treatment. (h) Western blot analysis of total kidney lysates from each group demonstrated decreased expression of ICAM-1 in IR-injured kidneys by exo-srIκB treatment. Comparisons between groups were assessed using one-way ANOVA with Bonferroni post-hoc test. Data are presented as mean ± SD values. ns; not significant, **P<0.01, ***P<0.001. When comparing PBS-sham and exo-naive-IRI surgery groups. #P<0.05, ##P<0.01, ###P<0.001 when comparing exo-naive IRI and exo-srIκB-IRI surgery groups. (1) Engineered exosome generation and characterization. (A) Schematic of DNA constructs used to produce super-repressor IκB-loaded exosomes (exo-srIκB) (top). Schematic showing fusion proteins and proposed activities (bottom). (B) Morphological characterization of exo-naive and exo-srIκB through transmission electron microscopy (TEM). (C) HEK293T cells stably expressing mCherry or srIκB and exosomes from these HEK293T cells were lysed and immunoblotted for the indicated proteins. (2) Protective effect of exo-srIκB in endotoxemia and cecal ligation and perforation (CLP)-induced sepsis. (A) Survival curves of exo-naive and exo-srIκB-treated septic mice treated with phosphate buffered saline (PBS). Lipopolysaccharide (LPS) C57BL/6 mice (n=5-6/group), LPS BALB/c mice (n=10/group) and CLP C57BL/6 mice (n=14-15/group). **p<0.01, *p<0.05 compared to PBS-treated septic group. (B) Levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and CCL4/macrophage inflammatory protein (MIP)-1β were measured in plasma of exosome-treated mice 24 hours after LPS injection or CLP. **p<0.01, *p<0.05 compared to PBS-treated sepsis group. †p<0.05 compared to Exo-naive-treated sepsis group. (C) Representative images of cortical duct cells of kidney sections from sham, CLP with PBS, CLP with Exo-naive, and CLP with Exo-srIκB, normal brush border (*) or brush border loss (○) in proximal ducts; chromatin condensation (white arrow); exposed basement membrane (white arrowhead); vacuolation (yellow arrow); scale bar, 100 μM. (D) Pathological kidney damage score of representative kidney samples from each group. *p<0.05 compared to PBS-treated sepsis group. †p<0.05 compared to Exo-naive-treated sepsis group. (3) Exosome biodistribution in LPS-injected mice. (A) In vivo imaging of mCLING-labeled exosome (red) uptake within neutrophils (LysM ^gfp/+ , green; Ly6G ⁺ , blue) in the liver of sham mice. (B) Representative time-lapse imaging of mCLING-labeled exosomes (red) within hepatic Ly6G ⁺ neutrophil cells (green) in LPS-treated C57BL/6 mice. (C) Stepwise images of the flow of mCLING-labeled exosomes (red) within the spleen of sham and LPS-treated LysM ^GFP/+ mice. Time lapse is indicated. Magenta, autofluorescence; scale bar, 50 μm. (4) Inhibitory effect of exo-srIκB on nuclear factor kappa B (NF-κB) signaling in vitro. (A) HEK293-NF-κB-luciferase cells (2×10 ⁴ cells) were cultured with exo-naive or exo-srIκB 2×10 ⁵ particles. After 24 h, cells were treated with 0.5 ng/ml TNF-α for an additional 18 h. Luciferase activity was measured and normalized. (B) Exo-srIκB dose-dependently inhibited NF-κB activation from NF-κB-luciferase cells. (C) THP-1 cells (5×10 ⁵ cells) were stimulated with 1 μg/ml LPS and treated with exo-srIκB 5×10 ⁶ particles. Supernatants were collected and assayed for production of TNF-α and monocyte chemoattractant protein (MCP)-1. JSH-23 (50 μM) was used as a positive control. **p<0.01. (D) Immunofluorescence of human umbilical vein endothelial cells (HUVECs) cultured with mCLING-tagged exo-srIκB. Representative images are presented. Nuclei were labeled with Hoechst. (E) HUVECs were stimulated with 300 ng/ml LPS for 24 h. Cells were harvested in single cell suspensions and assessed via flow cytometry using a specific phycoerythrin (PE)-conjugated antibody against human intercellular cell adhesion molecule (ICAM)-1. (S1) Exosome characterization. (A) Schematic of the procedure used to generate engineered exosomes. (B) Representative graphs of nanoparticle tracking analysis (NTA) demonstrating similar size distributions in diluted samples of exo-naive, exo-mCherry and exo-srIκB. (S2) Inhibitory effect of exo-srIκB in a mouse model of sepsis. (A) Body temperature changes were monitored after injection of the indicated exosomes into wild-type and LPS-treated C57BL/6 mice. **p<0.01 compared to the PBS-treated LPS group. (B) Representative images of kidney cortical regions in sections from C57BL/6 (upper panel) and BALB/c (lower panel) mice treated with LPS and PBS (left), exo-naive (middle) and exo-srIκB (right). Normal brush border (*) or brush border loss (○) in proximal ducts; chromatin condensation (red arrow); exposed basement membrane (red arrow); vacuolation (blue arrow); scale bar, 100 μM. (C) Ratio of the number of infiltrated Ly6G ⁺ cells in each organ between the exo-naive and exo-srIκB-treated groups of LPS-induced septic mice. (D) IL-10 levels were measured in the plasma of PBS-, exo-naive-, and exo-srIκB-treated septic mice. NS, not significant. (S3) Biodistribution of exosomes in mice with or without LPS challenge. (A) Analysis of mCLING-labeled exosomes detected from kidneys using an in vivo imaging system after intravenous administration of LPS. (S4) Inhibitory effect of exo-srIκB on LPS-induced inflammatory response in HUVEC. (A) HUVEC (7× ¹⁰⁴ cells) were cultured with 1× ¹⁰⁷ particles of exo-srIκB for 24 h, followed by stimulation with 36 ng/ml LPS. After 24 h, the supernatants were collected and analyzed for the production of IL-8 (left) and MCP-1 (right), respectively. JSH-23 (50 μM) was used as a positive control. ***p<0.001 compared to dimethyl sulfoxide (DMSO). #p<0.05 compared to cells treated with LPS alone. (B) HUVEC were pretreated with exo-srIκB for 24 h, followed by stimulation with 36 ng/ml LPS for 1 h. Protein levels of p65 and IκBα were assessed using Western blotting. GAPDH and histone H3 (HH3) were used as endogenous controls for cytoplasmic cell lysates and nuclear fractions, respectively (left). Binding of nuclear NF-κB to its target DNA sequence, 5'-GGGACTTTCC-3', was measured by enzyme-linked immunosorbent assay (ELISA) (right). Protective effect of exo-srIκB against lung injury in LPS-induced sepsis. H&E staining showing (1) alveoli; (2) capillaries; (3) neutrophils; (4) alveolar macrophages; (5) congestion; (6) hemorrhage; (7) necrosis. Protective effect of exo-srIκB against lung injury in LPS-induced sepsis. Survival curves of sham, naive exosome and exo-srIκB treated septic mice are shown. Protective effect of exo-srIκB against lung injury in LPS-induced sepsis. Levels of TNF-α in plasma of exo-srIκB-treated septic mice are shown. Protective effect of exo-srIκB against lung injury in LPS-induced sepsis. (1) Central vein and (2) H&E staining showing immune cell infiltration.

This disclosure provides the EXPLOR technology [see Yim et al., Nature Communications 7, 12277 (2016)], which was used to load srIκB into exosomes and deliver exo-srIκB systemically to evaluate their effects on the ischemic AKI process. Human embryonic kidney (HEK) 293T cell line, which produced two recombinant proteins, CIBN-EGFP-CD9 and srIκB-mCherry-CRY2, was used to produce srIκB-containing exosomes (exo-srIκB) by inducing the transient docking of CRY2 and CIBN using blue light illumination (Figure 1A). Control exosomes (exo-naive) were generated in intact HEK293T cells. The results confirmed that optogenetically engineered exosomes can be utilized as therapeutic agents and that exo-srIκB reduces IR-induced kidney damage through immune response regulation and apoptosis.

The singular terms ("a," "an," "the") and similar referents used in the context of describing this disclosure (particularly in the context of the claims) should be construed to include both the singular and the plural, unless otherwise expressly stated herein or contradicted by context. Ranges of values accepted herein are merely intended to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if each were individually referred to herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "for example") provided herein is merely to more fully describe the disclosure and does not limit the scope of the disclosure as specifically claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

As used herein, the term "about" refers to, for example, the length of a nucleotide sequence, the degree of error, the dimensions, the amount of a component in a composition, the concentration, the volume, the process temperature, the process time, the yield, the flow rate, the pressure, and similar values and variations in this range, and refers to variations in quantities that may occur, for example, through typical measuring and handling procedures used to manufacture a compound, composition, concentrate, or dosage form used; through inadvertent errors in these procedures; through differences in the manufacture, source, or purity of starting materials or components used to carry out these methods; and through other considerations. The term "about" also includes amounts that vary, for example, due to aging of a composition, dosage form, or cell culture having a particular initial concentration or mixture, and amounts that vary due to mixing or processing of a composition or dosage form having a particular initial concentration or mixture. The appended claims include equivalents to such amounts even when modified by the term "about". The term "about" can additionally refer to a range of values similar to the reference value mentioned. In certain embodiments, the term "about" refers to a range of values that are less than or equal to 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference value referenced.

As used herein, the term "subject" refers to a human or non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate).

AKI
Immune responses via NF-κB signaling can be involved in AKI, and exo-srIκB can improve kidney damage caused by kidney injury due to sepsis. In some examples of the present application, whether administration of an inhibitor can alleviate the process of ischemic AKI was evaluated. In the examples of the present application, srIκB was delivered to mice before and after IRI surgery using EXPLOR technology, and the results were compared with the control group. As a result, mice treated with exo-srIκB showed lower serum BUN and creatinine levels compared to the control group, thereby demonstrating the preventive and therapeutic effects against IR-induced AKI. Meanwhile, as a result of examining whether administration of exo-srIκB locally affects NF-κB signaling in the kidney, it was found that exo-srIκB significantly reduced the nuclear translocation and nuclear binding activity of NF-κB compared to the control group. The NF-κB pathway can play an important role in the immune response process. A comparison of gene expression of pan-inflammatory cytokines/chemokines between the Exo-srIκB treatment group and the control group, IL-1β, and IL-6 confirmed a significant decrease in multiple inflammatory mediators, including CCL2, CCL5, CXCL2, and TNF-α. Finally, in the present examples, flow cytometry was performed to compare whether treatment with an NF-κB inhibitor affected renal immune cell populations, and a significant decrease in the frequency of multiple immune cell populations, including neutrophils, monocytes/macrophages, and T cells, was observed.

IRI induces inflammatory cascades and oxidative stress, triggering a cytokine storm, inducing apoptosis and structural damage to adjacent tissues. The NF-κB signaling pathway can regulate cell survival and inflammation by a high level of rapid response from injured cells during IRI stimulation, especially during the early phase of hypoxia/reperfusion. A large number of endogenous factors, such as mobility group box 1 (HMGB1), heat shock proteins (HSPs), and pathogen/damage-associated molecular patterns (PAMPs/DAMPs), are released. These endogenous molecules can stimulate Toll-like receptors (TLRs) such as IL-1R and pattern recognition receptors (PRPs). TLRs and IL-1R share the same intracellular domain and phosphorylate inhibitory protein IκB residues, activating IκB kinase (IKK), which ultimately induces the degradation of IκB by the proteasome. This process allows heterodimers (e.g., p50/p65) to translocate from the cytoplasm to the nucleus, which binds to DNA and promotes the transcription of inflammatory mediators including TNF-α, IL-1, IL-6, and IL-8, accelerating NF-κB signaling. This proinflammatory cascade can regulate the surrounding microenvironment by inducing apoptosis and promoting leukocyte migration/activation in renal duct cells exposed to IRI. The ischemic AKI model of the present disclosure can also induce activation of NF-κB signaling, which induces the expression of multiple inflammatory cytokine/chemokine genes that significantly affect renal immune cell populations compared to the sham-operated group. Recent studies include TLR antagonists, cytokine antagonists, IKK complex antagonists, proteasome inhibitors, decoy oligodeoxynucleotides (ODNs) specific for specific NF-κB complexes, and ODNs that suppress Rel protein translation in various organs. This has the effect of blocking NF-κB with IRI by inhibiting NF-κB signaling using various pharmacological inhibitors. IκBα protein with mutations at serine residues 32 and 36 is not phosphorylated and therefore exists as a multimer with NF-κB in the cytoplasm and is not degraded, thus holding NF-κB nuclear translocation. This non-degradable IκBα, so-called super-repressor IκBα (srIκB), is known to induce apoptosis and exert antitumor effects.

In the present disclosure, the therapeutic effect of exo-srIκB is presented in a renal IRI model that represents acute nephritis. Exo-srIκB regulates the process of AKI by down-regulating the production of proinflammatory mediators that can limit leukocyte migration/activation and inducing early apoptosis of inflammatory cells to induce early termination of the inflammatory phase. In the present disclosure, it was confirmed that administration of exo-srIκB only affects the frequency of immune cell populations in the kidney, but not in the spleen. These results indicate that exo-srIκB selectively suppresses cell proliferation or promotes apoptosis in kidney-resident immune cells. Meanwhile, in vivo delivery of exo-srIκB can be interpreted as blocking the outflow of splenic immune cells to the injured kidney. In the present disclosure, exo-srIκB was found to attenuate IR-induced AKI in mice by suppressing the activity of NF-κB signaling. In addition, in vivo delivery of exo-srIκB can improve the overall inflammatory state of the kidney by reducing the expression of proinflammatory genes and decreasing renal immune cell populations including neutrophils, monocytes/macrophages, and T cells. Thus, in one aspect, the present disclosure relates to an exosome loaded with srIκB, and more particularly, to an exosome loaded with srIκB represented by the amino acid sequence of SEQ ID NO: 1. Hereinafter, srIκB may be referred to as a cargo protein.

In some embodiments, the AKI described herein is, but is not limited to, ischemia-reperfusion (IR)-induced AKI, lipopolysaccharide (LPS)-induced AKI, or sepsis-induced AKI. Ischemia-reperfusion (IR)-induced acute kidney injury (AKI) is a relatively common but serious medical condition that can occur when a subject suffers from systemic hypoperfusion followed by restoration of blood flow and reoxygenation. The most common clinical cases include patients undergoing kidney transplantation or cardiac surgery, and patients with sepsis, trauma, or myocardial infarction. Renal IR injury (IRI) is associated with high morbidity/mortality and is known to induce dysfunction in other distal organs including the heart, lungs, and liver. In IR-induced AKI, hypoxia and reperfusion generate reactive oxygen species (ROS), which then cause a cascade of reactions including apoptosis and inflammation, and subsequent renal failure. Immunologic responses can have a significant impact on renal IRI and repair. Nuclear factor (NF)-κB signaling, which regulates cytokine production and cell survival, is significantly involved in IR-induced AKI, and its inhibition can alleviate ischemic AKI.

Using EXPLOR, a surgically engineered exosome technology, exosomes were loaded with an exosomal super-repressor inhibitor of NF-κB (exo-srIκB) and administered to B6 WT mice before/after renal IR surgery as presented in the examples of this application. Results were compared to those of a control exosome (exo-naive) injected group. Exo-srIκB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in postischemic mice than the exo-naive treatment group. Systemic delivery of exo-srIκB reduced NF-κB activity and reduced levels of apoptosis in postischemic kidneys. Postischemic kidneys showed reduced gene expression of proinflammatory cytokines and adhesion molecules with exo-srIκB treatment compared to controls. Exo-srIκB treatment also significantly affected renal immune cell populations after ischemia, reducing the frequency of neutrophils, monocytes/macrophages, and T cells compared to the control group. Therefore, modulation of NF-κB signaling through exosome delivery can be used as a novel therapeutic approach for AKI, including IR-induced AKI.

In another embodiment of the invention, exosomes are engineered to contain a mutant form of a biological inhibitor of NF-κB called super-repressor (SR) IκB (srIκB, SEQ ID NO: 1) [see N. Yim et al., Nature Communications 7, 12277 (2016)].

As presented in the Examples below, an optogenetically engineered exosome system (EXPLOR) was used for loading large amounts of srIκB into exosomes in stably transfected cells. The Examples below confirm that intraperitoneal injection of purified srIκB-loaded exosomes (exo-srIκBs) attenuates mortality and systemic inflammation in a septic mouse model. In biodistribution studies using fluorescent dye-labeled exosomes, exo-srIκB was observed in mouse spleen and liver, primarily in neutrophils, and to a lesser extent in mononuclear cells. Furthermore, the Examples confirm that exo-srIκB attenuates inflammatory responses in mononuclear THP-1 cells and human umbilical vein endothelial cells (HUVECs) in response to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α. These results confirm that exo-srIκB can protect against systemic inflammation and sepsis-induced death by suppressing NF-κB activation.

The present disclosure provides a method of treating acute kidney injury (AKI) in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising an exosome comprising an NF-κB inhibitor. In some embodiments, the NF-κB inhibitor is selected from the group consisting of an NF-κB inhibitory protein, a fragment thereof, and a mixture thereof.

The composition is a physiologically acceptable aqueous solution or suspension of exosomes.

The NF-κB inhibitor is selected from an NF-κB inhibitory drug, an NF-κB inhibitory protein or a fragment thereof, or a mixture thereof.

Preferably, the NF-κB inhibitor is selected from the group consisting of super-repressor IκB, IκB-α, IκB-β, IκB-ε, BCL-3, mutants thereof, and mixtures thereof.

In yet another preferred embodiment of the present disclosure, the exosomes of the present disclosure contain a biological inhibitor of a mutant form of NF-κB called super-repressor (SR) IκB (srIκB, SEQ ID NO: 1).

In one embodiment of the present disclosure, srIκB has Ser32 and Ser36 of IκB (SEQ ID NO:2) substituted with Ala.

SEQ ID NO:1 Homo sapiens, super-repressor-IκB (srIκB)
MFQAAERPQEWAMEGPRDGLKKERLLDDRHDAGLDAMKDEEYEQMVKELQEIRLEPQEVPRGSEPWKQQLTEDGDSFLHLAIIHEEKALTMEVIRQVKGDLAFLNFQNNLQQTPLHLAVITNQPEIAEALLGAGCDPELRDFRGNTPLHLACEQGCLA SVGVLTQSCTTPHLHSILKATNYNGHTCLHLASIHGYLGIVELLVSLGADVNAQEPCNGRTALHLAVDLQNPDLVSLLLKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTESEFTEFTEDELPYDDCVFGGQRLTL

SEQ ID NO:2 Homo sapiens, IκB-α
MFQAAERPQEWAMEGPRDGLKKERLLDDRHDSGLDSMKDEEYEQMVKELQEIRLEPQEVPRGSEPWKQQLTEDGDSFLHLAIIHEEKALTMEVIRQVKGDLAFLNFQNNLQQTPLHLAVITNQPEIAEALLGAGCDPELRDFRGNTPLHLACEQGCLA SVGVLTQSCTTPHLHSILKATNYNGHTCLHLASIHGYLGIVELLVSLGADVNAQEPCNGRTALHLAVDLQNPDLVSLLLKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTESEFTEFTEDELPYDDCVFGGQRLTL

SEQ ID NO: 3 Recombinant srIκBα peptide DRHDAGLDAMKDE

In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:1. In embodiments, the NF-κB inhibitor is represented by SEQ ID NO:1.

In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:2. In some examples, the NF-κB inhibitor comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor is represented by SEQ ID NO:1. In some embodiments, the NF-κB inhibitor is represented by SEQ ID NO:2.

In certain embodiments of the present disclosure, the exosome further comprises a photo-specific binding protein. In some embodiments, the photo-specific binding protein comprises a first photo-specific binding protein and/or a second photo-specific binding protein that reversibly interact with each other upon irradiation. In some embodiments, the first photo-specific binding protein is conjugated to an exosome-specific marker to form a first fusion protein (fusion protein I). In some embodiments, the second photo-specific binding protein is conjugated to an NF-κB inhibitory protein to form a second fusion protein (fusion protein II). In some embodiments, the fusion protein I and the fusion protein II are reversibly linked through the first photo-specific binding protein and the second photo-specific binding protein. In some embodiments, the first photo-specific binding protein is conjugated to the exosome-specific marker to be located toward the inside of the exosome.

In additional embodiments, the first photospecific binding protein and the second photospecific binding protein are selected from the group including, but not limited to, CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

In additional embodiments, the first photospecific binding protein is CIB or CIBN and the second photospecific binding protein is CRY or PHR; or the first photospecific binding protein is CRY or PHR and the second photospecific binding protein is CIB or CIBN.

In additional embodiments, the first photospecific binding protein is PhyB and the second photospecific binding protein is PIF, or the first photospecific binding protein is PIF and the second photospecific binding protein is PhyB.

In additional embodiments, the first photospecific binding protein is GIGANTEA and the second photospecific binding protein is FKF1, or the first photospecific binding protein is FKF1 and the second photospecific binding protein is GIGANTEA.

In some embodiments, the exosome-specific marker is selected from the group including, but not limited to, CD9, CD63, CD81, or CD82. In additional embodiments, the exosome-specific marker is CD9. In additional embodiments, the exosome-specific marker is CD63. In additional embodiments, the exosome-specific marker is CD81. In additional embodiments, the exosome-specific marker is CD82.

In some embodiments, the exosomes have a diameter of about 50 nm to about 200 nm. In some embodiments, the exosomes have a diameter of about 50 nm to about 75 nm. In some embodiments, the exosomes have a diameter of about 75 nm to about 100 nm. In some embodiments, the exosomes have a diameter of about 100 nm to about 125 nm. In some embodiments, the exosomes have a diameter of about 125 nm to about 150 nm. In some embodiments, the exosomes have a diameter of about 150 nm to about 175 nm. In some embodiments, the exosomes have a diameter of about 175 nm to about 200 nm. In embodiments, the exosomes have a diameter of about 50 nm to about 150 nm. In some embodiments, the exosomes have a diameter of about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or more and/or a diameter of about 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or less.

Without describing all the details, the contents of U.S. Pat. No. 10,702,581 and Korean Pat. No. 10-2100420 are incorporated herein by reference to provide compositions and methods for producing exosomes containing the NF-κB inhibitors of the present disclosure.

The present disclosure also provides sustained release of a therapeutically active drug, whereas conventional sustained effect requires repeated administration of the therapeutic agent.

The present disclosure also provides a pharmaceutical composition comprising an extracellular vesicle (exosome) carrying an NF-κB inhibitor and a pharma- ceutically acceptable carrier or carriers.

Despite recent notable progress in understanding the pathophysiology of IR-induced AKI, no drugs with proven clinical efficacy and safety have been developed to treat ischemic AKI; therefore, the associated levels of mortality and morbidity are significant. Because NF-κB signaling is deeply involved in the process of IR-induced AKI, we evaluated whether systemic delivery of an NF-κB inhibitor using exosomes could attenuate the ischemic AKI process. Exo-srIκB was delivered to mice before and after IRI surgery through optically controlled exosome biogenesis using EXPLOR technology, and the results were compared with those of a control group. The results showed that the mice group provided with exo-srIκB treatment was protected from IR-induced AKI; it showed better biochemical and histological outcomes than the control group. Systemic delivery of exo-srIκB suppressed NF-κB signaling in the postischemic kidney, which reduced the expression of proinflammatory cytokines/chemokines and adhesion molecules and attenuated apoptosis. Finally, NF-κB treatment affected renal immune cell populations, with a significant decrease in the frequency of multiple immune cell populations, including neutrophils, monocytes/macrophages, and T cells, observed through flow cytometry and immunofluorescence analysis.

During the early process of IRI, proinflammatory cascades and oxidative stress lead to NF-κB pathway activation, which subsequently induces cell survival and regulation of inflammation. During the early stage of hypoxia/reperfusion, multiple endogenous factors including high mobility group box 1, heat shock proteins and pathogen/injury-associated molecular patterns are released, which stimulate pattern recognition receptors such as Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). This process subsequently activates IκB kinase (IKK) to induce IκB degradation by the proteasome. Subsequently, heterodimers (e.g. p50/p65) translocate from the cytosol to the nucleus, where they promote the transcription of inflammatory mediators including TNF-α, IL-1, IL-6 and IL-8, and further promote NF-κB signaling. These proinflammatory cascades regulate the surrounding microenvironment by inducing apoptosis in tubular cells exposed to IRI and promoting leukocyte migration/activation. This experimental ischemic AKI model can recapitulate the activation of NF-κB signaling after renal IRI, which can induce increased expression of multiple inflammatory cytokines/chemokines and tissue apoptosis that significantly affect renal immune cell populations.

Recent experimental studies have provided more detailed evidence of the effect of NF-κB blockade on IRI using selective pharmacological inhibitors of NF-κB signaling, including TLR antagonists, cytokine antagonists (33), IKK complex antagonists, proteasome inhibitors, and decoy oligodeoxynucleotides specific for certain NF-κB complexes in various organs. srIκB is a non-degradable IκBα protein with mutations at serine residues 32 and 36. This protein prevents NF-κB nuclear translocation and subsequent NF-κB signaling. srIκB has been shown to have protective effects in pulmonary IRI models by reducing neutrophil infiltration and pulmonary edema, and is also known to exert antitumor effects by reducing chemoresistance. However, the effect of srIκB treatment during the course of kidney injury has not been fully evaluated. Using bioengineered exosomes as vectors, Choi et al. recently demonstrated the therapeutic benefit of srIκB treatment in a septic AKI model. The following examples also demonstrate the protective effect of exo-srIκB treatment in a renal IRI model, extending the therapeutic potential of srIκB delivery through exosomes in various types of AKI.

Modulation of the inflammatory process has been an important therapeutic consideration in the field of ischemic AKI. Splenectomy and the use of the anti-TNF-α agent, infliximab, reduced the expression of inflammatory cytokines and the accumulation of macrophages/monocytes, exerting a protective effect in experimental ischemic AKI models. Inhibiting leukocyte and macrophage migration preserved kidney function and reduced cell death. The examples provided showed that exo-srIκB treatment down-regulated the expression of proinflammatory cytokines/chemokines and adhesion molecules, reduced apoptosis, and reduced the accumulation of multilineage immune cells in the IR-injured kidney. These results suggest that exo-srIκB transforms the course of ischemic AKI through down-regulation of the proinflammatory cascade, limiting subsequent leukocyte migration/activation, and preventing apoptosis.

Exosomes have several advantages over other types of vectors used as conveyors. First, EVs are naturally protected from degradation in the circulation and are non-immunogenic when used autologously. Exosomes can also overcome natural barriers such as the blood-brain barrier through their inherent cell targeting properties. Furthermore, exosomes use recipient cell-intrinsic mechanisms during uptake, intracellular transport and final delivery of cargo to target cells. While maintaining these properties, the EXPLOR technology could significantly increase the intracellular levels of cargo proteins through controllable reversible detachment from exosomes. Intracellular delivery of therapeutic proteins has several technical obstacles, including low purification efficiency, induction of immune responses against the host and limited cytosolic delivery, and this novel exosome delivery method using the EXPLOR technology has significantly improved exosome loading performance, delivery efficiency and purity compared to those of previous technologies. The following examples show that efficient delivery of exo-srIκB using the EXPLOR technology leads to significant improvement in renal outcomes after experimental IRI.

Systemic exo-srIκB treatment only affects the frequency of immune cell populations in the kidney but not in the spleen. This may involve exo-srIκB acting selectively on kidney-resident immune cells to reduce proliferation or promote apoptosis, or systemic delivery of exo-srIκB to block the outflow of splenic immune cells to the injured kidney. Kidney-resident immune cells and immune cells from lymphoid organs are known to have distinct origins, phenotypes, and functional properties. Additional studies using chimeric mice or in vivo imaging could distinguish resident kidney immune cells from circulating immune cells and provide more information on the immune cell populations affected by systemic exo-srIκB treatment.

Using exosomes as carriers to deliver active therapeutic cargo is a safe and efficient method to deliver srIκB in IR-induced AKI models. Exo-srIκB treatment attenuates IR-induced AKI in mice by downregulating NF-κB signaling and ameliorating inflammation and apoptosis in ischemic damaged kidneys. Direct intracellular delivery of immunosuppressive proteins to target cells using exosomes is a promising therapeutic tool for IR-induced AKI, and the therapeutic potential of exo-srIκB in human renal IRI remains to be further explored.

Sepsis-Induced Diseases Sepsis is accompanied by multiple organ failure and excessive inflammatory responses, and has complex pathophysiological characteristics, making it difficult to develop a therapeutic agent that can effectively treat all symptoms. Sepsis is initiated through the strong activation of the innate immune system and mediated by the activation of pattern recognition receptors (PRRs) by pathogens, inducing activation of the complement system, coagulation system, and vascular endothelium, making normal homeostasis difficult.

As presented in the examples of the present application, the effect of exo-srIκB was able to resolve inflammatory responses and reduce organ damage through the inhibition of NF-κB activity in a septic mouse model. srIκB is a non-degradable form of IκBα that prevents NF-κB from entering the nucleus and acting as a transcription factor. For example, recent techniques that can load functional proteins into exosomes were utilized to generate srIκB-loaded immunosuppressive exosomes. By systemically delivering exo-srIκB in a septic mouse model (e.g., systemic delivery), the examples of the present application confirmed that exosomes were delivered to neutrophils and macrophages, which are key participants in the septic inflammatory response. Also, in both mouse sepsis models of the present example, exo-srIκB treatment significantly reduced pro-inflammatory markers such as TNF-α, IL-1β, and IL-6, but not the anti-inflammatory cytokine IL-10. A significant therapeutic effect of exo-srIκB was observed even when exosomes were injected 1 hour after LPS injection. Therefore, treatment with exo-srIκB may be applicable to the early stage of sepsis before the transition to an immunosuppressive state.

Proinflammatory cytokines can play an important role in sepsis-induced acute kidney injury (AKI). In the examples of this application, in a mouse model with AKI induced by CLP surgery or LPS injection, histopathological examination confirmed that glomerular structure was destroyed, tubular epithelial cells were degenerated, and severe intracellular edema and congestion occurred in the tubules of the sepsis-induced group. However, exo-srIκB administration reduced the severity of the lesions, kidney damage, and infiltration of inflammatory cells.

Neutrophil tissue infiltration may be a key process in sepsis. For example, some studies have reported that targeting specific adhesion molecules to deplete neutrophils and inhibit neutrophil recruitment can reduce tissue damage and protect in sepsis. TNF-α and IL-1β are major mediators for the expression of chemokines such as MCP-1 and IL-8, which are important chemoattractants for neutrophils and monocytes/macrophages during inflammatory responses. Such chemokines initiate local infiltration of monocytes/macrophages after activation. However, in HUVECs, exo-srIκB was shown to reduce LPS-induced release of MCP-1 and IL-8, thereby inhibiting monocyte/macrophage infiltration. Such data are consistent with the results obtained in the in vivo study of the present application. In this example, exo-srIκB treatment significantly reduced neutrophil infiltration in the spleen, kidney, lung, and liver of a sepsis animal model. Considering the important role of neutrophils in the pathophysiology of sepsis, the tissue protective effect of exo-srIκB may be associated with a reduction in neutrophil infiltration, at least in the kidney.

In some embodiments, the spleen exhibits high levels of neutrophils compared to other organs such as the kidney and liver. Proinflammatory cytokines may be generated in the spleen and may affect other organs. Most genes encoding proinflammatory cytokines may be under the regulation of NF-κB, one of the key transcription factors in the septic inflammatory response induced by LPS. The expression of NF-κB-responsive reporter proteins in the presence or absence of exo-srIκB treatment was investigated in the present examples, and translocation of p65 was confirmed. Exo-srIκB suppressed NF-κB signal activation by blocking the nuclear translocation of NF-κB subunit 65.

In some embodiments, exosomes are used as a mechanism for therapeutic delivery of immunosuppressive proteins in a mouse model of sepsis. Exosomes are excellent carriers for the delivery of srIκB to cells, providing a new option for the treatment of sepsis. Although a method for directly delivering immunosuppressive proteins to target cells in vivo has not previously been established, exo-srIκB according to the present disclosure acts as an inhibitor of NF-κB, thereby suppressing inflammation-related gene expression and direct inflammatory responses. Examples of the present application can suppress this to reduce the proinflammatory cytokine storm and resulting organ damage in sepsis.

In animal models of sepsis induced by either lipopolysaccharide (LPS; endotoxin model) or cecal ligation and perforation (CLP; polymicrobial model), recent studies have demonstrated that NF-κB inhibitors with diverse chemical properties and mechanisms of action protect animals from septic lethality. Over 700 NF-κB inhibitors have been reported, but to date no NF-κB blockers have been approved for human use. A variety of steroidal and nonsteroidal anti-inflammatory drugs have been shown to block NF-κB, but their effects are highly pleiotropic and lack specificity. Novel therapeutic strategies aimed at specific inhibition of key elements in the NF-κB activation pathway have recently been developed, with high hopes for their potential efficacy as a treatment for sepsis and for one or more diseases induced by sepsis. A genetic construct expressing an engineered IκBα protein lacking phosphorylation sites, super-repressor IκB (srIκB), has also been used. Mutation of IκBα at specific phosphorylation sites (Ser32 and Ser36 replaced by Ala) results in a dominantly active form of IκBα with an extended half-life. Such srIκB induces a stable cytoplasmic pool of IκBα, thereby preventing nuclear NF-κB activation.

Here, exosomes were used as therapeutic conveyors to deliver srIκB to therapeutic targets. Exosomes have been recognized as powerful therapeutic mediators to transfer diverse proteins and regulatory genes to target cells. They function as non-immunogenic nanovesicles and can protect their cargo from serum proteases and immune responses. Loading soluble proteins into exosomes is made possible by a technology called EXPLOR, which uses the natural exosome biogenesis process and reversible protein-protein interactions controlled by optogenetics. To load specific target proteins into exosomes, a HEK293T cell line was generated that stably expresses two recombinant proteins, CIBN-EGFP-CD9 and srIκB-mCherry-CRY2 (Figure 1A). Consistent with previous studies, the EXPLOR technology was used to produce srIκB-loaded exosomes. srIκB-loaded exosomes were designated as exo-srIκB, and exosomes generated from uninjured HEK293T cells were designated as exo-naive. Therapeutic application of exo-srIκB to a sepsis model ameliorated sepsis-induced organ damage and suppressed the secretion of proinflammatory cytokines, thereby improving the overall survival rate of septic patients.

The present disclosure provides a method for treating sepsis-induced disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising exosomes that contain an NF-κB inhibitor.

In embodiments, the disease is selected from the group consisting of, but not limited to, pneumonia, cytokine storm syndrome, respiratory distress syndrome, and organ failure. In some embodiments, the disease is pneumonia. In some embodiments, the disease is cytokine storm syndrome. In some embodiments, the disease is respiratory distress syndrome. In some embodiments, the disease is organ failure.

In an embodiment, the NF-κB inhibitor is selected from the group consisting of NF-κB inhibitory proteins, fragments thereof, and mixtures thereof. In an embodiment, the NF-κB inhibitor is selected from the group consisting of super-repressor (SR)-IκB (srIκB), IκB-α, IκB-β, IκB-ε, BCL-3, mutants thereof, and mixtures thereof. In an embodiment, the NF-κB inhibitor is super-repressor (SR)-IκB (srIκB).

In embodiments, the composition is administered via oral, transdermal, intraperitoneal, intravenous, intramuscular, subcutaneous, or mixed routes. In some embodiments, the composition is administered via oral route. In some embodiments, the composition is administered via transdermal route. In some embodiments, the composition is administered via intraperitoneal route. In some embodiments, the composition is administered via intravenous route. In some embodiments, the composition is administered via intramuscular route. In some embodiments, the composition is administered via subcutaneous route. In some embodiments, the composition is administered via mixed route.

In an embodiment, the method further includes administering an anti-inflammatory agent to the subject.

In embodiments, the exosome comprising the NF-κB inhibitor further comprises a photospecific binding protein. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the NF-κB inhibitor is represented by SEQ ID NO:1.

In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:2. In some embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2. In embodiments, the NF-κB inhibitor is represented by SEQ ID NO:1. In some embodiments, the NF-κB inhibitor is represented by SEQ ID NO:2.

In some embodiments, the photospecific binding protein is a first photospecific binding protein and/or a second photospecific binding protein. In embodiments, the first photospecific binding protein is conjugated to an exosome-specific marker to form fusion protein I; the second photospecific binding protein is conjugated to an NF-κB inhibitor to form fusion protein II. In embodiments, fusion protein I and fusion protein II are reversibly linked through the first photospecific binding protein and the second photospecific binding protein. In embodiments, the first photospecific binding protein is conjugated to an exosome-specific marker such that it is located in a direction toward the inside of the exosome.

In some embodiments, the first light-specific binding protein and the second light-specific binding protein are selected from the group consisting of CIB, CIBN, PhyB, PIF FKF1, GIGANTEA, CRY, and PHR. In some embodiments, the first light-specific binding protein is CIB and the second light-specific binding protein is CRY. In some embodiments, the first light-specific binding protein is CIBN and the second light-specific binding protein is CRY. In some embodiments, the first light-specific binding protein is CIB and the second light-specific binding protein is PHR. In some embodiments, the first light-specific binding protein is CIBN and the second light-specific binding protein is PHR.

In some embodiments, the first light-specific binding protein is CRY or PHR and the second light-specific binding protein is CIB or CIBN. In some embodiments, the first light-specific binding protein is CRY and the second light-specific binding protein is CIB. In some embodiments, the first light-specific binding protein is CRY and the second light-specific binding protein is CIBN. In some embodiments, the first light-specific binding protein is PHR and the second light-specific binding protein is CIB. In some embodiments, the first light-specific binding protein is PHR and the second light-specific binding protein is CIBN.

In some embodiments, the first photospecific binding protein is PhyB and the second photospecific binding protein is PIF. In some embodiments, the first photospecific binding protein is PIF and the second photospecific binding protein is PhyB.

In some embodiments, the first photospecific binding protein is GIGANTEA and the second photospecific binding protein is FKF1. In some embodiments, the first photospecific binding protein is FKF1 and the second photospecific binding protein is GIGANTEA.

In some embodiments, the exosome-specific marker is selected from the group consisting of CD9, CD63, CD81, and CD82. In some embodiments, the exosome-specific marker is CD9. In some embodiments, the exosome-specific marker is CD63. In some embodiments, the exosome-specific marker is CD81. In some embodiments, the exosome-specific marker is CD82.

In some embodiments, the exosomes have a diameter of about 50 nm to about 200 nm. In some embodiments, the exosomes have a diameter of about 50 nm to about 75 nm. In some embodiments, the exosomes have a diameter of about 75 nm to about 100 nm. In some embodiments, the exosomes have a diameter of about 100 nm to about 125 nm. In some embodiments, the exosomes have a diameter of about 125 nm to about 150 nm. In some embodiments, the exosomes have a diameter of about 150 nm to about 175 nm. In some embodiments, the exosomes have a diameter of about 175 nm to about 200 mm. In embodiments, the exosomes have a diameter of about 50 nm to about 150 nm. In some embodiments, the exosomes have a diameter of about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 150 or more and/or a diameter of about 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or less.

In some embodiments, the composition is a pharmaceutical composition further comprising a physiologically acceptable carrier or carriers.

EXPLOR® Technology Recently, exosomes have received significant attention as novel biocarriers for gene/drug delivery. Exosomes are extracellular vesicles (EVs) that play an important role in intercellular communication by delivering bioactive substances to recipient cells and influencing the signaling pathways of target cells. Exosomes are easier to store and exhibit greater stability. Exosomes have a high dose that can overcome biological barriers and can carry surface molecules that target specific cell types, thus resulting in fewer off-target effects. However, in the clinical application of exosomes, obtaining a large amount of pure exosomes and loading soluble proteins into exosomes have been significant challenges. Using a novel optogenetically engineered exosome technology developed by Yim et al., “Exosomes for Protein Loading via Photoreversible Protein-Protein Interaction” (EXPLOR), significant progress has been made in the production efficiency and biological compatibility of exosomes. This EXPLOR technology was recently adopted by Choi et al. in an experimental sepsis model; they delivered exosomes containing super-repressor IκB (srIκB) to mice, a non-degradable form of kappa B (IκB) inhibitor that prevents nuclear translocation of NF-κB. These studies showed that exo-srIκB treatment ameliorated the systemic inflammatory response and subsequent organ dysfunction in a mouse sepsis model.

EXAMPLES Animals: Male C57BL/6J mice were housed under specific pathogen-free conditions in the central animal facility of Yonsei Medical Center. Male mice aged 6-7 weeks were used for all experiments. C56BL/6, C57BL/6N, and BALB/c mice were purchased from Orientbio (Seongnam, Republic of Korea). LysM ^GFP/+ mice, which constitutively express GFP in neutrophils and macrophages, were generously provided by Dr. M. Kim (University of Rochester, Rochester, NY, USA).

Exosome isolation: srIκB-loaded exosomes were produced using a HEK293T cell line expressing two recombinant proteins, CIBN-EGFP-CD9 and srIκB-mCherry-CRY2. Intact HEK293T cells were used for exo-naive production. Stable cells were seeded in T175 flasks for 24 hours at 37°C. After removing the medium, the cells were washed and resuspended in exosome-depleted medium. The cells were then exposed to continuous blue light illumination from a 460 nm light-emitting diode in a _CO2 incubator. After 72 hours, the cell culture supernatant was collected, centrifuged to remove cells and debris, and filtered through a 0.22 μm polyethersulfone filter; exosomes were isolated using a combination of tangential flow filtration and size exclusion chromatography. In some examples, stable cells were seeded in T175 flasks to produce exo-srIκB. After 1 day, the medium was carefully removed, the cells were washed with PBS, and exosome-depleted medium was added. The cells were then exposed to continuous blue light illumination from a 460 nm light-emitting diode in a _CO2 incubator. After 72 hours, the cell culture supernatant was collected and centrifuged at 1,000 g for 15 minutes to remove cells and cell debris, and then filtered through a 0.22 μm polyethersulfone (PES) filter to remove large particles. Exosomes were isolated using molecular weight cut-off (MWCO)-based membrane filtration. The isolated exosomes were purified by SEC.

Transmission electron microscopy: To observe the morphology of EVs through TEM, 5 μL of EVs were absorbed onto carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA) for 15 seconds. After removing excess liquid, samples were negatively stained with 2% uranyl acetate (Electron Microscopy Sciences). TEM images were obtained using a Tecnai G2 Retrofit electron microscope (FEI company Hillsboro, OR) operating at 200 kV. Extracellular vesicles (EVs) were morphologically evaluated through negative staining. First, 5 μl of EVs suspended in PBS were loaded onto glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA). After 3-5 seconds of sample adsorption, the grids were blotted with filter paper and stained with 2% uranyl acetate (UA). The samples were then dried for 20 seconds using a dryer. EVs were observed with a Tecnai G2 Retrofit (FEI Company, Hillsboro, OR, USA) at a voltage of 200 kV.

Nanoparticle tracking analysis: The particle number and size distribution of EVs were measured by NTA using a Zetaview instrument (PMX120, Particle Metrix Germany) equipped with a 488 nm laser. Samples were diluted in particle-free PBS according to the manufacturer's instructions (serum 1:100 to 1:10,000). Samples were analyzed at 25°C under constant flow conditions with camera level 78 and shutter 80. The size distribution determined by NTA corresponds to the hydrodynamic diameter of EVs in suspension. The particle number was calculated from the Brownian motion velocity, and the size was determined using the two-dimensional Stokes-Einstein equation based on the particle motion velocity. Certain samples were diluted 1:100 to 1:10,000 with 0.2 μM filtered PBS. EV concentration was measured based on 50 to 200 particle numbers per frame. For each measurement, two cycles of scanning at 11 cell positions were performed with the following settings: focus, autofocus; camera sensitivity for all samples, 78.0; shutter, 70; and cell temperature, 25°C. EV concentrations were expressed as particles per ml (pn/ml).

Immunoblotting: Cells were lysed in RIPA buffer containing Halt® protease and phosphatase inhibitor cocktail (100X) (Thermo Fisher Scientific, Waltham, MA) and kidneys were lysed in Triton X-100 lysis buffer. Lysates were centrifuged at 9,000-16,000 x g for 10-20 min at 4°C and the supernatants were stored at -70°C for subsequent immunoblotting. Protein concentrations were measured using a BCA assay (Pierce® BCA Protein Assay, Thermo Scientific TM). Protein extracts were dissolved in Laemmli sample buffer (Bio-Rad, Hercules, CA) and heated at 100°C for 5 min. Proteins were electrophoresed on acrylamide-denaturing SDS-polyacrylamide gels and transferred to nitrocellulose (NC) membranes. The transfer membrane was incubated in blocking buffer A (PBS, 0.1% Tween-20, and 5% nonfat milk) at 22° C. for 1 hour, and then incubated with a primary antibody against srIκB (customized antibody, Abelon, Seoul, South Korea), mCherry, Alix, TSG101, GM130, calnexin (Abeam Cambridge, UK), CD9, CD63 (SBI, Tokyo, Japan), GAPDH (Santa Cruz Biotechnology, Dallas, TX), prohibitin (NOVUSBIO, Centennial, CO), nucleoporin p62 (BD bioscience, San Diego, CA), and IgG1 (IgG1). The cells were incubated overnight at 4°C with 5% CO, 10% ethanol, 10% sucrose ... After washing and incubation of the membranes with specific secondary antibodies, they were developed with chemiluminescent agents [Clarity and Clarity Max ECL Western Blotting Substrates (Bio-Rad) or West-Q Pico Dura ECL solution (GenDEPOT, Katy, TX)]. The relative optical density of the bands for each group was measured using Image J software (NIH).

Mouse renal ischemia-reperfusion model: Mice were anesthetized via intraperitoneal administration of a tiletamine/zolazepam (Zoletil®)-xylazine (Rompun®) mixture (1 mg/kg) and positioned on a heated surgical pad (37°C). Bilateral IRI was performed using a dorsal approach. Renal pedicles were dissected and microclamps (Jeungdo Bio & Plant, Seoul, Republic of Korea) were placed on each renal pedicle for 30 min. Sham animals received anesthesia and bilateral dorsal incisions alone. During surgery, mice were hydrated with 1 ml of warm normal saline to prevent dehydration. After 30 min of ischemia, both clamps were removed and blood flow recovery was confirmed before closing the wound with the Autoclip® wound closure system (Harvard apparatus, Holliston, MA). The animals were allowed to recover with free access to food and water. Mice were sacrificed 24 or 48 hours after IRI surgery. Serum, spleen, and kidneys were collected from each mouse for further analysis. Serum BUN and creatinine values were analyzed using a Cobas C502 autoanalyzer (Roche diagnostics, Basel, Switzerland).

Enzyme-linked immunosorbent assay (ELISA): Serum NGAL levels were measured using a mouse ELISA kit according to the manufacturer's protocol (Mouse Lipocalin-2/NGAL Quantitative ELISA Kit) (R&D Systems Minneapolis, MN) after diluting samples 1:100-1000 times.

TNF-α, IL-8, and MCP-1 levels in supernatants collected from THP-1 and HUVEC cultures were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN USA), and NF-κB activity in HUVEC was measured using the TransAM NF-κB p65 kit (Active Motif, Carlsbad, CA, USA). TNF-α, CCL-4/macrophage inflammatory protein (MIP)-1β, IL-6, and IL-1β levels in mouse plasma collected via cardiac puncture at the indicated time points after the advanced CLP procedure were measured using a mouse Magnetic Luminex eScreening Assay kit (R&D Systems). Assays were performed according to the manufacturer's instructions. All samples and standards were assayed in duplicate using the Luminex 200™ system (Merck Millipore, Darmstadt, Germany). To measure serum TNF-α, IL-1β, IL-6, and CCL4 at the indicated time points, blood samples were collected via cardiac puncture after the LPS endotoxemia procedure, allowed to clot for 2 h at room temperature, and subsequently centrifuged at 2,000 g for 20 min at 4°C.

NE-PER nuclear and cytoplasmic extraction: Kidneys were collected, cut into small pieces, washed with PBS, and bead-homogenized with a specific volume of cytoplasmic extraction reagent (CER) 1 from the NE-PER nuclear and cytoplasmic extraction reagent kit (ThermoFisher, Waltham, MA) according to the weight of the kidney. After 15 s of vigorous vortexing and 10 min of incubation on ice, CER II was added to each kidney sample, followed by 1 min of incubation on ice and centrifugation at 16,000 × g for 5 min. The supernatant was collected with the cytoplasmic extract and the insoluble fraction was suspended in the nuclear extraction reagent. After 10 min of centrifugation at 16,000 × g, all the supernatant was collected and used as the nuclear extract.

Histopathological assessment of kidney injury score and immunohistochemistry: Five-micrometer-thick sections of formalin-fixed and paraffin-embedded kidney tissue samples were processed through hematoxylin-eosin (HE) and IHC staining. To histologically assess kidney injury, each slide was stained with HE. A renal pathologist (JIY) blindly scored the ratio of necrotic ducts to total ducts in the cortical region. Kidney injury was scored according to the ratio of damaged ducts to total ducts: 0, normal; 1, <25% damage; 2, 25-50% damage; 3, 50-75% damage; 4, 75-90% damage; and 5, >90% damage. Histological criteria for damaged ducts included loss of ductal brush border, vacuolation, chromatin condensation, and exposed ductal basement membrane. Generally, tubules were evaluated and scored in five randomly selected high-powered fields containing 50-100 ducts.

For IHC, tissue sections were deparaffinized, rehydrated with ethyl alcohol, and washed with tap water. Antigens were retrieved in 10 mM sodium citrate buffer for 20 min using a Black & Decker vegetable steamer. Slides were blocked with 10% donkey serum for 30 min at 22°C and washed with PBS. Primary antibodies against NF-κB (Clone 8242S, Cell signalling technology, Danvers, MA) and ICAM-1 (Clone AF796, R&D Systems) were diluted to appropriate concentrations in 2% casein in bovine serum albumin, added to the slides, and incubated overnight at 4°C. After washing, secondary antibodies (Dako, Carpinteria, CA) were applied for 1 h at 22°C. Diaminobenzidine was added for 2 min, and slides were counterstained with hematoxylin. Semiquantitative scores of staining intensity were obtained by examining at least five fields in each section through digital image analysis (MetaMorph version 4.6r5, Universal Imaging Corp., Downingtown, PA) under 400x magnification.

Measurement of DNA binding activity of NF-κB: Using 20 μg of kidney nuclear extract, the binding activity of p65/c-Rel (NF-κB) to DNA was measured using the TransAM NF-κB Assay Kit (ActiveMotif, Carlsbad, CA) according to the manufacturer's protocol.

Quantitative real-time polymerase chain reaction: A well-established method for whole kidney RNA extraction was used. Briefly, whole kidney samples were homogenized with 700 μl of RNAiso Reagent (Takara Bio Inc., Otsu, Shiga, Japan). 160 μl of chloroform was then added to the homogenized samples of kidney cells. The mixture was then vigorously shaken for 30 s, maintained at 22°C for 3 min, and centrifuged at 16,000×g for 15 min at 4°C. The aqueous phase located at the top of the three phases was carefully transferred to a fresh tube to prevent contamination with other phases. The extracted RNA was precipitated by adding 400 μl of isopropanol and centrifuged at 16,000×g for 30 min at 4°C. The RNA pellet was washed with 70% ethanol, air-dried for 2 min, and dissolved in sterile diethylpyrocarbonate-treated distilled water. The quantity and quality of the extracted RNA was assessed spectrophotometrically at wavelengths of 260 and 280 nm. The isolated RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Takara Bio Inc, Shiga Prefecture, Japan). 5 μl of each DNA was mixed with 10 μl of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 5 pmol of sense/antisense primers. Primer concentrations were determined after preliminary experiments to confirm the optimal concentration for each primer. The expression levels of IL-1α, IL-1β, Il-2, Il-4, Il-6, Il-10, Il-17α, Ccl2, Ccl3, Ccl5, Cxcl2, Ifn-γ, Tnf-α, and Icam-1 were measured using an ABI PRISM 7700 sequence detection system (Applied Biosystems). The PCR conditions were as follows: an initial heating step was performed at 95°C for 9 min, followed by 35 cycles of denaturation at 94.5°C for 30 min, annealing at 60°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The sequence data of each primer are presented in Table 1.

Each sample was run in triplicate in separate tubes, and a negative control without cDNA was run in parallel with each assay. After real-time PCR, the temperature was increased from 60°C to 95°C at a rate of 2°C/min to generate melting curves. Expression values for each gene were normalized to the value of 18S rRNA, and relative fold expression values were calculated using the comparative C _T method with 2 ^{−△△C T} .

Multiplex cytokine assay in serum: Thirteen inflammatory cytokines and chemokines, including IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, CCL2, CCL3, CCL5, CXCL2, IFN-γ, and TNF-α, were analyzed using the Milliplex Mouse Cytokine/Chemokine Magnetic bead Panel multiplex assay kit according to the manufacturer's protocol (EMD Millipore Billerica, MA) using 1:2 diluted serum. Quantification was performed using a Luminex 200 microplate reader (EMD Millipore).

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay: Cell death was assessed by FITC-labeled TUNEL assay using a commercial kit (S7110, Merck, Millipore, Billerica, MA). TUNEL-positive cells in formalin-fixed kidney tissues were identified by examining a minimum of five fields of each section with digital image analysis (MetaMorph version 4.6r5, Universal Imaging Corp.) at 400x magnification.

Isolation of renal mononuclear cells and splenocytes from mice: KMNCs were isolated by established protocols. Both kidneys were removed after exsanguination, decapsulated, finely minced, and incubated with collagenase type I (1 mg/ml) (Worthington Biochemical Lakewood, NJ) for 30 min at 37°C. After enzymatic digestion, single cell kidney suspensions were achieved by mechanical disruption of the tissue through a 70 μm strainer (BDbioscience, San Jose, CA). KMNCs were collected after centrifugation at RT using an isotonic Percoll density gradient (GE Healthcare, Chicago, IL) (1,500 × g for 30 min in brake-off mode) according to the manufacturer's instructions. The collected cells were washed, resuspended in RPMI medium containing 5% FBS, and counted automatically using a Countess® II FL automated cell counter (Life Technologies, Waltham, MA).

Spleen cells were collected through mechanical disruption of the spleen using a 70 μm strainer (BD bioscience) and incubated with RBC lysis solution (Qiagen, Hilden, Germany) for 3 min to remove red blood cells.

FACS sorting of kidney CD4 ⁺ T cells: For FACS sorting, KMNCs were preincubated with anti-CD16/CD32 Fc block (clone 93, Biolegend, San Diego, CA) for 10 min on ice and stained with monoclonal Abs anti-CD45 (APC, Clone 30-F11, Biolegend), anti-Ly6G (APC-Fire 750, Clone 1A8, Biolegend), anti-Ly6C (FITC, Clone HK1.4, Biolegend), anti-F4/80 (PerCP-Cy5.5, Clone BM8, Biolegend), and anti-CD3 (PE, Clone 17A2, Biolegend) for 30 min at 4°C. After washing and resuspension in FACS buffer (PBS with 5% FBS), stained cells were analyzed on an LSRII using FACS Diva software (BD Biosciences) and FlowJo software (version 10.2). After removing debris/dead cells and doublets, each sample was analyzed based on the expression of CD45, Ly6G, Ly6C, F4/80 and CD3. OneComp eBeads (ThermoFisher) were used for compensation.

Immunofluorescence: After deparaffinization, rehydration and antigen retrieval, slides were blocked with 10% chlorinated serum for 30 min at 22°C and washed with Tween-20 in phosphate-buffered saline. Primary antibodies against Ly-6G (Clone ab25377, Abcam, Cambridge, UK), Ly-6C (Clone ab15627, Abcam), F4/80 (Clone ab6640, Abcam) and fluorescein (FITC)-conjugated Lotus tetragonolobus lectin (LTL) (Clone FL-1321 Vector laboratories, Burlingame, CA) were diluted to appropriate concentrations in 2% casein in bovine serum albumin, added to the slides and incubated overnight at 4°C. After washing, goat anti-rat IgG Alexa Fluor 647 (ab150167, Abcam) was applied for 1 h at 22°C. Slides were then counterstained with 4',6-diamidino-2-phenylindole (DAPI) (MBD0015, Sigma-Aldrich, St. Louis, MO). Staining intensity was scored semiquantitatively by examining a minimum of five fields in each section at 400x magnification via digital image analysis (MetaMorph version 4.6r5, Universal Imaging Corp.).

Statistics: Data were expressed as mean ± standard deviation (SD) values. Statistical differences were analyzed using one-way ANOVA with Bonferroni post-test using Prism 8 (GraphPad, San Diego, CA). Results indicated statistically significant differences when P < 0.05.

LPS endotoxemia model: To generate a mouse model of LPS endotoxemia, male mice were injected with LPS derived from Escherichia coli (Sigma-Aldrich, Milwaukee, WI, USA).

High-grade CLP sepsis model and treatment regimen: Male C57BL/6 mice purchased from ORIENTBIO (Seongnam-si, Gyeonggi-do, Korea) were submitted to high-grade CLP at 9-10 weeks of age using the procedure previously described with minor modifications. All mice were housed and experiments were performed in a specific pathogen-free area of the Medical Research Institute, Yonsei University College of Medicine, approved by the Association for Assessment and Accreditation of Laboratory Animal Care International. Mice were anesthetized via ip injection of a mixture of 80 mg/kg ketamine and 10 mg/kg xylazine before all procedures. After anesthesia, the peritoneum was opened in a sterile manner, and the cecum was ligated at the distal end with 1 cm of 4-0 black silk thread and perforated with a 23 gauge needle. After perforation, the needle was removed and a small amount of stool was pushed out through both perforations to ensure patency. The cecum was then placed back into the abdominal cavity, the abdominal incision was closed with 6-0 nylon suture, and stainless steel removable wound clips were applied to the skin. After this procedure, 1 ml of pre-warmed saline per 20 g body weight was administered subcutaneously. Sham-operated mice underwent the same procedure except for the ligation and perforation of the cecum. After this procedure, 1.0 x ¹⁰⁹ particles of exo-naive or exo-srIκB were administered to the mice via ip injection at 0, 6, 12, and 18 hours. As a control group, the same volume of phosphate buffered saline (PBS) was injected in the same manner. Animals were evaluated every 2 hours for the first 48 hours after CLP, followed by every 4 hours for 5 days. Samples were collected to evaluate the outcome of the procedure within 18 hours after CLP.

Intravital imaging: A laser scanning intravital confocal microscope (IVM-C; IVIM Technology, Daejeon, Republic of Korea) was used to visualize the biodistribution and anti-corruption effect of exo-srIκB. During intravital imaging, the body temperature of the mice was maintained at 37°C using a homoeothermic controller. The mice were anesthetized with an intramuscular injection of zoletil (30 mg/kg) and xylazine (10 mg/kg). Small incisions of 10 mm were made in both the skin and peritoneum to access the internal organs, including the liver, spleen, and kidneys. The exposed organs were kept hydrated by repeated application of saline during imaging. Exosome biodistribution was visualized through wide-area z-stack imaging before and after exo-naive or exo-srIκB injection. To fluorescently label neutrophils in C57BL/6N mice in vivo, anti-Ly6G antibody (BD Bioscience, San Jose, CA, USA) conjugated with Alexa Fluor 555 (A20009; Thermo Fisher Scientific, Waltham, MA, USA) was injected intravenously. Naive or srIκB exosomes labeled with mCLING ATTO 647N (Synaptic System, Göttingen, Germany) were injected intravenously through the tail vein catheter or a 31-G insulin syringe. To generate a sepsis model, high-dose LPS (Sigma-Aldrich, St. Louis, MO, USA) was injected intravenously 1 to 16 hours before imaging.

Confocal microscopy: For exosome uptake analysis, exosomes were diluted in 0.5 ml of Dulbecco's PBS (DPBS). The suspension was then incubated with 647N-labeled mCLING-ATTO (Synaptic Systems) according to the manufacturer's instructions. The mixture was then pelleted through centrifugation, and 300 μl of the resuspended pellet was incubated with HUVECs. After 24 h, cells were washed with DPBS and fixed in 4% paraformaldehyde solution. Hoechst was used for nuclear staining. Images were recorded using a Zeiss 710 confocal microscope (Zeiss Oberkochen, Germany).

Luciferase assay: HEK293 cells were stably transfected with a luciferase receptor construct regulated under the NF-κB response element (SL-0012; Signosis, Santa Clara, CA, USA) and grown according to the manufacturer's instructions. Cells were treated with exosomes for 24 h at 37°C, and luciferase activity was measured (E1501; Promega, Madison, WI, USA) using a SpectraMax ID3 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Flow cytometry: The levels of surface markers expressed on HUVECs were assessed using flow cytometry. Cells were detached, harvested via centrifugation, and labeled with a phycoerythrin (PE)-conjugated antibody specific for human ICAM-1 (BD Biosciences) for 30 minutes on ice in the dark, followed by extensive washing. All samples were analyzed on a BD Celesta flow cytometer (BD Biosciences). Data were analyzed using BD FACSDiva software. A PE-conjugated antibody specific for IgGlk (BD Biosciences) was used as an isoform control.

Example 1. Characterization and Analysis of Engineered Exosomes Exosome production, harvesting and purification have been fully described in previous studies, as shown in FIG. 13. The particle size was mostly 30-120 nm, with an average size of 101 nm. Transmission electron microscopy (TEM) showed intact cup-shaped membrane vesicles with sizes according to nanoparticle tracking analysis (NTA) results (FIG. 1, B and C). Immunoblotting analysis results for exo-srIκB showed strong expression of target proteins including srIκB, mCherry, CD9 and GFP with positive exosomal markers such as CD63, TSG101, Alix and GAPDH. Exo-srIκB lacked expression of organelle markers including prohibitin, calnexin, GM130 and nucleoporin 62. Exo-naive did not express any markers except exosomal markers (FIG. 1D).

Example 2. Injection of exo-srIκB ameliorates renal ischemia-reperfusion injury.
First, we investigated the role of exo-srIκB during renal IRI. Each experimental group was intraperitoneally injected three times with 3×10 ⁹ pn exo-srIκB or exo-naive at 1-h intervals (total of 9×10 ⁹ pn) before and after renal IRI (pre-treatment: 3, 2, and 1 h before IRI surgery, post-treatment: 1, 2, and 3 h after IRI surgery). Mice were sacrificed 24 or 48 h after surgery (Figure 2A).

Interestingly, the mice receiving exo-srIκB showed significantly lower serum blood urea nitrogen (BUN) and creatinine levels after IRI surgery than the exo-naive injection group in both pre- and post-treatment models (24-h/48-h BUN and creatinine in both models, P<0.001) (Figures 2B and 2C). Serum neutrophil gelatinase-associated lipocalin (NGAL) levels were significantly decreased in the exo-srIκB injection group in the pre- and post-treatment models compared to the exo-naive group (P<0.001) (Figure 2D). Histological evaluation results also showed lower vascular damage scores in the mice receiving exo-srIκB treatment compared to the exo-naive group in the pre/post-treatment model (P<0.05 vs. exo-naive injection group) (Figure 2E). Collectively, these data indicate that systemic delivery of exo-srIκB directly prevents the progression of ischemic AKI.

Example 3. Systemic exo-sIκB treatment suppresses renal NF-κB signaling in IR-injured kidneys.
To understand the underlying mechanism by which systemic exo-srIκB treatment affects the IR-induced AKI process, we first investigated the systemic delivery of exo-srIκB to determine whether it inhibits local NF-κB signaling in the kidney. The expression of NF-κB p65 protein in kidney nuclear extracts from each different experimental group was measured through Western blotting. Systemic treatment with 9 × 10 ⁹ pn exo-srIκB reduced IR-induced NF-κB nuclear translocation in the pre-IRI (IRI 24 h, P <0.05; 48 h, P < 0.01) and post-IRI (IRI 24-/48 h, P < 0.01) (Figure 3A) treatment models compared with that found in the exo-naive treatment group. This finding was reconfirmed by measuring the DNA binding activity of p65 using kidney nuclear extracts from each experimental group. Exo-srIκB treatment significantly down-regulated the DNA binding activity of NF-κB after renal IRI compared with the exo-naive group, regardless of the treatment time point (pre-treatment: 24-/48-h IRI, P<0.05; post-treatment: 2-/48-h IRI, P<0.01) (Fig. 3B). Further validation through NF-κB immunohistochemistry (IHC) staining showed that the expression of NF-κB was significantly decreased in the exo-srIκB-treated group compared with the control-treated group (pre-treatment: 24-h IRI, P<0.05; 48-h, P<0.001 and post-treatment: 24-/48-h IRI, P<0.05) (Fig. 3C). Thus, systemic exo-srIκB treatment can reduce renal NF-κB signaling in the kidney after IR.

Example 4. Exo-srIκB ameliorates post-ischemic renal inflammation.
To further investigate whether systemic exosomal srIκB treatment locally attenuates renal IRI-induced inflammation, gene expression levels of central inflammatory mediators, including IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17α, Ccl2, Ccl3, Ccl5, Cxcl2, Ifn-γ, and Tnf-α, were determined through quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with 9×10 ⁹ pn of Exo-TIκB prior to IRI significantly suppressed the expression levels of IL-1β, IL-6, Tnf-α, Ccl2, Ccl5, and Cxcl2 in IR-injured kidneys compared with those of the Ex-naive treated group ( FIG. 4A ). Multiplex cytokine analysis was also performed using sera from different experimental groups. Although the results were not as significant as the renal transcriptional data, there was a clear trend toward decreased expression of proinflammatory cytokines, including IL-6, TNF-α, CCL2, CCL5, and CXCL2, in the exo-srIκB-treated group compared with that in the control group (Figure 4B).

Furthermore, the effect of exosomal srIκB treatment on adhesion molecule expression was evaluated by comparing the levels of transcribed/translated intracellular adhesion molecule 1 (ICAM-1) among treatment groups. QRT-PCR results showed significantly lower levels of Icam-1 expression in exo-srIκB-treated groups than exo-naive treatment in both pre- and post-treatment models (Figure 4C). This result was reproduced at the translational level through Western blotting and IHC staining (Figure 4, D and E). Thus, systemic exo-srIκB signaling can down-regulate the expression of proinflammatory cytokines/chemokines and adhesion molecules in IR-induced AKI.

Example 5. Exo-srIκB reduces apoptosis in the kidney after ischemia.
Based on the known role of NF-κB in regulating programmed cell death, we investigated how exo-srIκB affected apoptosis in the postischemic kidney. Renal IRI surgery induced significant apoptosis in kidney cells, which was shown by a sharp increase in the levels of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) in Western blot analysis. Systemic delivery of exo-srIκB before/after injury could substantially down-regulate the expression levels of cleaved caspase-3 and cleaved PARP, suggesting that exo-srIκB exerts a protective effect against apoptosis in the postischemic kidney (Figure 5A). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to determine the extent of kidney cell injury. Compared with the control group, exo-srIκB-treated kidneys showed significantly fewer TUNEL-positive cells in the pre/post treatment model (Figure 5B).

Example 6. Exosome biodistribution after renal ischemia-reperfusion injury The biodistribution of exo-srIκB in a renal IRI model was investigated to better understand which cell types modulate the protective effect of exo-srIκB in the postischemic kidney. Mice were intravenously injected with fluorochrome-labeled Ly6G and F4/80 antibodies 1 hour before IRI surgery and intravenously injected with ^9x109 pn exo-srIκB 1 hour after reperfusion. Intravital imaging was performed at 10 min, 5 hours, and 24 hours after reperfusion (Figure 6A). Intravital imaging after DiD-labeled exosome injection, as well as anti-Ly6G antibody (Fluor 555) to visualize neutrophils and anti-F4/80 antibody (Fluor 488) to visualize macrophages, confirmed that both exo-srIκB and exo-naive were taken up by neutrophils (Ly6G ⁺ ) and macrophages (F4/80 ⁺ ) in the postischemic kidney (FIG. 6B). We observed a slight increase in DiD (green) signal in the tubules after IRI, enhancing the possibility of exosome uptake by tubular cells, but the signal intensity was not significantly different from that before DiD-labeled exosome injection (data not shown). The spleen was observed simultaneously, which showed exosome uptake by neutrophils and macrophages in the outer parenchymal region (FIG. 6C). The inner parenchymal region showed no significant uptake (data not shown).

Example 7. Systemic delivery of exo-srIκB affects renal immune cell populations after renal IRI Immune cells are known to play an important role in the process of ischemic AKI; therefore, we evaluated whether providing exosomal srIκB treatment prior to IRI surgery affects the population of each immune cell type. Twenty-four hours after ischemic AKI, there was no difference in the total number of kidney cells isolated through multiple steps of mechanical disruption, enzymatic digestion, and Percoll density gradient methods between the experimental groups, but the exo-srIκB-treated group had a significantly lower frequency of kidney mononuclear cells (KMNCs) than that of the exo-Niave-treated group (P<0.05) (FIG. 7, A and B). Additional analysis showed that the exo-srIκB-injected group had significantly lower frequencies of neutrophils (CD45 ⁺ Ly6G ⁺ ) (P < 0.01), pro-/anti-inflammatory mononuclear phagocytes (CD45 ⁺ Ly6C ⁺ /CD45 ⁺ F4/80 ⁺ ) (P < 0.01/P < 0.05, respectively), and T cells (CD45 ⁺ CD3 ⁺ ) (P < 0.05) in total KMNC than those of the exo-naive-injected group (Figure 7C). Immunofluorescence results of IR-injured kidneys also showed decreased neutrophil and mononuclear phagocyte frequencies in exo-srIκB-injected kidneys (Figure 7D). However, exo-srIκB treatment did not significantly affect the total immune cell numbers or frequencies in the spleen after ischemic AKI. There were no differences in the total cell numbers and frequencies of total immune cells (CD45 ⁺ ), neutrophils (CD45 ⁺ Ly6G ⁺ ), and pro/anti-inflammatory mononuclear phagocytes (CD45 ⁺ Ly6C ⁺ /CD45 ⁺ F4/80 ⁺ ), except for T cells (CD45 ⁺ CD3 ⁺ ), between the exo-srIκB and exo-naive treated groups, confirming that systemic exo-srIκB treatment prior to ischemic AKI has a significant local effect on the proliferation/trafficking of renal immune cells but not on splenic immune cells.

Example 8. Intravenous Delivery of Exo-srIκB To verify whether similar protective effects as those of intraperitoneal exosome delivery could be found with intravenous delivery, some experiments were repeated using an intravenous injection method. The improved biochemical results, as well as the reduced levels of NF-κB signaling and ICAM-1 expression by intravenous delivery of Exo-srIκB, were reproduced as shown in FIG. 8. Thus, intravenous delivery of Exo-srIκB results in similar biological effects compared to intraperitoneal delivery in the ischemic AKI model.

Example 9. Further characterization and analysis of srIκB-loaded exosomes Culture medium was collected from each cell type to produce sufficient exosomes.

HUVECs were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in F-12K medium (ATCC) containing 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Worth, CO, USA), heparin (Sigma-Aldrich), endothelial cell growth supplement (BD Biosciences), and 1% penicillin/streptomycin (Thermo Fisher). HUVECs between the 3rd and 6th passages were used for all experiments. Human mononuclear cells (THP-1) were purchased from ATCC and cultured in RPMI 1640 medium (Welgene Daegu Korea) supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich).

Exosomes were isolated using a combination of tangential flow filtration (TFF) and size exclusion chromatography (SEC) (Figure 13A). To reduce the risk of impurities exceeding the binding capacity being loaded onto the SEC column, the sample was diafiltered and concentrated through TFF. After TFF, the concentrated medium was loaded onto the SEC column for further purification. A second TFF was then performed to concentrate the exosomes. Each type of exosome isolated through stepwise purification was analyzed through nanoparticle tracking analysis (NTA), thereby characterizing the size and concentration of exosomes (Figure 13B). Particles with a diameter of 30-120 nm accounted for more than 80% of all particles, with an average size of 101 nm, which is consistent with the characteristic size range of exosomes (30-120 nm). Transmission electron microscopy (TEM) also showed intact cup-shaped membrane vesicles with sizes corresponding to the NTA results obtained from exo-naive and exo-srIκB (Figure 9B). To further characterize the exosomes in the formulations of the present invention, two common exosome markers were investigated. These were the tetraspanins CD63 and TSG101 through Western blotting.

Western blotting was performed as described in previous studies. Antibodies targeting the following proteins were used: IκBα (CST4812; Cell Signaling Technology, Danvers, MA, USA), p65 (CST6956S; Cell Signaling Technology), CD9 (NBP2-22187; NOVUSBIO, Centennial, CO, USA), CD63 (EXOAB-CD63A-1; SBI, Tokyo, Japan), TSG101 (ab228013; Abcam, Cambridge, UK), GM130 (ab52649; Abcam), GAPDH (sc-47724; Santa Cruz), and IL-11 (IL-11). Biotechnology, Santa Cruz, CA, USA), Histone H3 (ab1791; Abcam), mCherry (ab125096; Abcam), and GFP (CST2555; Cell Signaling Technology).

The presence of CD63 and TSG101 was clearly observed in the samples, while the bone fat-derived contaminant GM130 was only detected in the cell lysates (Figure 9C). Such analysis of exosome preparations indicates that they have exosomal properties. We also observed robust loading of srIκB-mCherry-CRY2 (130 kDa) in exosomes isolated from srIκB stable cell lines.

Example 10. Exo-srIκB improves survival and alleviates acute organ damage in septic mice We investigated whether intraperitoneal (i.p.) injection of exo-srIκB has a protective effect against endotoxic shock. Animals were given a single i.p. injection of a lethal dose of LPS (40 mg/kg body weight for C57BL/6 and 20 mg/kg body weight for BALB/c) followed by a single i.p. injection of exo-srIκB (6 h apart). Compared to control C57BL/6 mice, which had a 100% mortality rate from LPS-induced sepsis, mice treated with exo-srIκB were significantly resistant to LPS-induced mortality; the majority of mice were rescued from sepsis and had prolonged survival (Figure 10A, top). Exo-srIκB-mediated effects were associated with modest protection from LPS-induced temperature drop (Fig. 14A). A mouse model of LPS-induced sepsis was developed using BALB/c mice. Exo-srIκB treatment significantly improved the survival rate of LPS-induced sepsis in BALB/c mice (Fig. 10A, middle). We subsequently investigated the role of exo-srIκB in survival using a CLP-induced sepsis model in a standardized mouse model of abdominal sepsis. Animals survived for a monitoring period of 7 days, indicating that exo-srIκB provides sustained protection against sepsis mortality (Fig. 10A, bottom). To further investigate whether exo-srIκB treatment attenuates sepsis-induced inflammation, the concentrations of key inflammatory factors were determined in serum using enzyme-linked immunosorbent assays (ELISAs). It was observed that the expression levels of the proinflammatory cytokines TNFα, IL-1β, and IL-6, and the chemokine carbon tetrachloride (CCL4) were significantly decreased in the exo-srIκB-treated group with LPS-induced sepsis (Fig. 10B, top). In addition, the serum levels of TNF-α and CCL4 in the CLP group were significantly higher than those in the sham-operated control mice, but were not elevated in the mice treated with either CLP or exo-srIκB (Fig. 10B, bottom). These results suggest that exo-srIκB attenuates inflammation induced by LPS or CLP and protects septic mice from acute inflammatory processes.

Numerous studies have revealed that acute kidney injury (AKI) is a frequent and serious complication of sepsis, occurring in more than 50% of sepsis cases and resulting in a very high mortality rate. Renal histology was performed to determine the role of exo-srIκB in CLP-induced AKI in a septic mouse model. Compared with the sham-operated group, tubular cells of phosphate-buffered saline (PBS)-treated and exo-naive mice showed substantial damage associated with vacuolation, loss of brush border, and nuclear condensation of ductal epithelial cells (Fig. 10C). Notably, exo-srIκB treatment significantly reduced damage to the ductal site and preserved the proximal duct, demonstrating the therapeutic potential of exo-srIκB against sepsis-induced kidney injury. These results are consistent with those of LPS-induced AKI (Fig. 14B). Further quantification of the severity of histological kidney injury showed that exo-srIκB treatment significantly reduced the number of injury points by approximately 50% in both sepsis models (Figure 10D). Exo-srIκB improved the degenerative changes in tubules, even though some damaged areas were found. These data suggest the critical importance of exo-srIκB in improving renal physiological structure and function in septic mice.

Example 11. Exosome biodistribution after LPS injection We investigated the changes in exosome biodistribution in an LPS-injected septic mouse model. In vivo biodistribution was analyzed using a custom-made intravital video-rate laser scanning confocal microscopy system. mCLING fluorescent dye-labeled exosomes were intravenously injected into LysM ^gfp/+ transgenic C57BL/6 mice that had previously been injected with anti-Ly6G antibodies to visualize neutrophils (green fluorescent protein [GFP] and Alexa 555) and macrophages (GFP). We observed that most exosomes were rapidly taken up by neutrophils and macrophages, i.e., within a few minutes after intravenous exosome injection (FIG. 11A).

Exosomes were primarily delivered to neutrophils in the liver (Fig. 11B), and increased recruitment and exosome uptake of neutrophils and macrophages was observed in the spleen of LPS-induced mice (Fig. 11C). mCLING-labeled exosomes were also delivered to neutrophils in the kidney of LPS-injected mice (Fig. 15A). These observations suggest that therapeutic exosomes were successfully delivered to target neutrophils and macrophages within 30 minutes in a septic mouse model.

Example 12. Exo-srIκB Attenuates Sepsis-Associated Inflammation We confirmed the target specificity and efficiency of exo-srIκB in vitro. Exo-srIκB, but not exo-naive, significantly blocked TNF-α-induced NF-κB activation in HEK293 cells stably expressing an NF-κB luciferase reporter gene (FIG. 12A). Increasing the amount of exo-srIκB reduced NF-κB reporter activity in a dose-dependent manner, indicating that exosome-released srIκB prevents NF-κB transcriptional activity (FIG. 12B).

Among all sepsis-reactive cells, monocytes/macrophages play the most important role in promoting immune responses, and depletion of these cells in septic mice increases mortality. THP-1 cells are widely used as monocytes in cell culture models. When THP-1 cells were stimulated with LPS, treatment with exo-srIκB reduced the secretion of the proinflammatory cytokines TNF-α and monocyte chemotactic protein (MCP)-1, which are under NF-κB transcriptional regulation, compared with exo-naive cells (Figure 12C). The effect of exo-srIκB was also compared with that of JSH-23, a commonly used NF-κB inhibitor that interferes with NF-κB target DNA binding. Exemplary data showed that exo-srIκB and JSH-23 exerted comparable effects in suppressing LPS-induced NF-κB activation and cytokine production in THP-1 cells (Figure 12C, lane 4 vs. lane 6).

Sepsis is characterized by mononuclear cell adhesion to the endothelium. Under normal conditions, endothelial cells are quiescent and do not interact with mononuclear cells. However, in an inflammatory environment, activated endothelial cells express adhesion molecules such as intercellular cell adhesion molecule (ICAM)-1, which bind to mononuclear cells. It is widely assumed that adhesion molecule expression is activated during the development of sepsis.

To study the effect of exo-srIκB on the expression of cell adhesion molecules in human umbilical vein endothelial cells (HUVEC), the present example first investigated whether exo-srIκBs are internalized into cells. mCLING-tagged exo-srIκB was detected in HUVEC, with maximum internalization observed after 24 hours (Figure 12D). We also confirmed that ICAM-1 expression in HUVEC was significantly suppressed by exo-srIκB treatment (Figure 12E). IL-8 and MCP-1 also induced the migration and adhesion of monocytes to the vascular endothelium and induced these cells to extravasate in the surrounding tissues. Expression of IL-8 and MCP-1 has previously been observed in various tissues during acute inflammation due to bacterial and viral infections and is associated with severe sepsis. Exo-srIκB suppressed LPS-induced IL-8 and MCP-1 production (Fig. 16A) by suppressing the transcriptional activity of NF-κB in HUVECs (Fig. 16B). These results confirm that exo-srIκB reduces NF-κB-mediated inflammatory responses in sepsis.

Example 13. Protective effect of exo-srIκB against lung injury in LPS-induced sepsis Animals received a single intraperitoneal injection of a lethal dose of LPS (30 mg/kg body weight) for 30 min, followed by a single intravenous injection of exo-srIκB (1×10 ¹⁰ ) for 10 h. Lung tissues were analyzed by H&E staining as presented in FIG. 17. The group injected with LPS for 10 h showed neutrophil infiltration and proliferation of alveolar macrophages, intra- and extra-alveolar congestion, and hemorrhage progressed. Alveolar endothelial cell damage, as well as alveolar bone necrosis, were observed. Exosome administration reduced alveolar wall expansion, neutrophil infiltration, and alveolar macrophage proliferation, even when some congestion was observed.

30 mg/kg LPS was administered to the LPS-treated group (negative control group) and the 3-dose exo-srIκB-treated group, consisting of 10 mice per group. 30 min after administration, exo-srIκB was administered once i.v. and survival rates were assessed for 72 h as presented in Figure 18. Compared to control C57BL/6 mice, mice treated with high doses of exo-srIκB were resistant to LPS-induced mortality and had prolonged survival rates.

30 mg/kg LPS was administered to the LPS-treated group (negative control group) and the 3-dose exo-srIκB-treated group, consisting of 5 mice per group. 30 min after administration, exo-srIκB was administered once i.v. and plasma was obtained at each time point by necropsy. The resulting plasma levels of TNF-α were measured by ELISA (Figure 19). The expression level of the proinflammatory cytokine TNF-α was decreased in a time-dependent manner in the exo-srIκB-treated group with LPS-induced sepsis.

The animals received a single intraperitoneal injection of a lethal dose of LPS (30 mg/kg body weight) for 30 min, followed by a single intravenous injection of exo-srIκB (1×10 ¹⁰ ). Liver tissue was analyzed by H&E staining ( FIG. 20 ). One hour after LPS administration, slight infiltration of Kupffer and immune cells around the central vein was observed, while infiltration of inflammatory cells was reduced in the exosome-administered group. Ten hours after LPS administration, infiltration of immune cells around Kupffer and the central vein was significantly increased, and connective tissue damage between hepatocytes was observed. Although infiltration was reduced in the exosome-administered group, the necrotic and edema process of hepatocytes was expected to be gradually alleviated. Therefore, repeated administration of exosomes is expected to enhance the protective effect.

Embodiments for Treating AKI Embodiment 1. A method for treating acute kidney injury (AKI) in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising exosomes comprising an NF-κB inhibitor.

Embodiment 2. In any one of the preceding embodiments, the method wherein the NF-κB inhibitor is selected from the group consisting of NF-κB inhibitory proteins, fragments thereof, and mixtures thereof.

Embodiment 3. In any one of the preceding embodiments, the NF-κB inhibitor is selected from the group consisting of super-repressor (SR)-IκB (srIκB), IκB-α, IκB-β, IκB-ε, BCL-3, mutants thereof, and mixtures thereof.

Embodiment 4. In any one of the preceding embodiments, the method wherein the NF-κB inhibitor is super-repressor (SR)-IκB (srIκB).

Embodiment 5. In any one of the preceding embodiments, the composition is administered orally, transdermally, intraperitoneally, intravenously, intramuscularly, subcutaneously, or via a mixed route.

Embodiment 6. The method of any one of the preceding embodiments, wherein the subject is a human.

Embodiment 7. The method of any one of the preceding embodiments, wherein the method further comprises administering an anti-inflammatory agent to the subject.

Embodiment 8. The method of any one of the preceding embodiments, wherein the exosomes containing the NF-κB inhibitor further contain a photospecific binding protein.

Embodiment 9. The method of any one of the preceding embodiments, wherein the NF-κB inhibitor comprises SEQ ID NO:1 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1.

Embodiment 10. In any one of the preceding embodiments, the NF-κB inhibitor is represented by SEQ ID NO: 1.

Embodiment 11. In any one of the preceding embodiments, the NF-κB inhibitor is represented by SEQ ID NO:2.

Embodiment 12. In any one of the preceding embodiments, the photospecific binding protein is a first photospecific binding protein and/or a second photospecific binding protein; the first photospecific binding protein is conjugated to an exosome-specific marker to form fusion protein I; and the second photospecific binding protein is conjugated to an NF-κB inhibitor to form fusion protein II.

Embodiment 13. The method of embodiment 12, wherein fusion protein I and fusion protein II are reversibly bound via a first photospecific binding protein and a second photospecific binding protein.

Embodiment 14. The method of embodiment 12 or 13, wherein the first photospecific binding protein is conjugated to an exosome-specific marker so as to be positioned in a direction toward the inside of the exosome.

Embodiment 15. In any one of embodiments 12 to 14, the first photospecific binding protein and the second photospecific binding protein are selected from the group consisting of CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, and PHR.

Embodiment 16. The method of embodiment 15, wherein the first photospecific binding protein is CIB or CIBN, and the second photospecific binding protein is CRY or PHR.

Embodiment 17. The method of embodiment 15, wherein the first photospecific binding protein is CRY or PHR, and the second photospecific binding protein is CIB or CIBN.

Embodiment 18. The method of embodiment 15, wherein the first photospecific binding protein is PhyB and the second photospecific binding protein is PIF.

Embodiment 19. The method of embodiment 15, wherein the first photospecific binding protein is PIF and the second photospecific binding protein is PhyB.

Embodiment 20. The method of embodiment 15, wherein the first photospecific binding protein is GIGANTEA and the second photospecific binding protein is FKF1.

Embodiment 21. The method of embodiment 15, wherein the first photospecific binding protein is FKF1 and the second photospecific binding protein is GIGANTEA.

Embodiment 22. The method of any one of embodiments 12 to 21, wherein the exosome-specific marker is selected from the group consisting of CD9, CD63, CD81, and CDS2.

Embodiment 23. In any one of the preceding embodiments, the exosomes have a diameter of about 50 nm to about 200 nm.

Embodiment 24. In any one of the preceding embodiments, the exosomes have a diameter of about 50 nm to about 150 nm.

Embodiment 25. The method of any one of the preceding embodiments, wherein the composition is a pharmaceutical composition comprising a physiologically acceptable carrier.

Embodiment 26. A composition comprising an exosome further comprising an NF-κB inhibitor as described in embodiments 1 to 16 for treating AKI.

Embodiment 27. Use of a composition comprising an exosome containing an NF-κB inhibitor as described in embodiments 1 to 16 for treating AKI.

Embodiments for Treating Other Diseases Embodiment 1. A method of treating sepsis induced disease in a subject in need thereof, comprising administering to said subject an effective amount of a composition comprising exosomes comprising an NF-κB inhibitor.

Embodiment 2. The method of any one of the preceding embodiments, wherein the disease is selected from the group consisting of pneumonia, cytokine storm syndrome, respiratory distress syndrome, and organ failure. In some embodiments, the disease is pneumonia. In some embodiments, the disease is cytokine storm syndrome. In some embodiments, the disease is respiratory distress syndrome. In some embodiments, the disease is organ failure.

Embodiment 3. In any one of the preceding embodiments, the NF-κB inhibitor is selected from the group consisting of NF-κB inhibitory proteins, fragments thereof, and mixtures thereof.

Embodiment 4. In any one of the preceding embodiments, the NF-κB inhibitor is selected from the group consisting of super-repressor (SR)-IκB (srIκB), IκB-α, IκB-β, IκB-ε, BCL-3, mutants thereof, and mixtures thereof.

Embodiment 5. In any one of the preceding embodiments, the NF-κB inhibitor is super-repressor (SR)-IκB (srIκB).

Embodiment 6. In any one of the preceding embodiments, the composition is administered orally, transdermally, intraperitoneally, intravenously, intramuscularly, subcutaneously, or via a mixed route.

Embodiment 7. The method of any one of the preceding embodiments, wherein the subject is a human.

Embodiment 8. The method of any one of the preceding embodiments, wherein the method further comprises administering an anti-inflammatory agent to the subject.

Embodiment 9. The method of any one of the preceding embodiments, wherein the exosomes containing the NF-κB inhibitor further contain a photospecific binding protein.

Embodiment 10. In any one of the preceding embodiments, the NF-κB inhibitor comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

Embodiment 11. The method of embodiment 10, wherein the NF-κB inhibitor is represented by SEQ ID NO: 1.

Embodiment 12. The method of embodiment 10, wherein the NF-κB inhibitor is represented by SEQ ID NO:2.

Embodiment 13. In any one of the preceding embodiments, the photospecific binding protein is a first photospecific binding protein and/or a second photospecific binding protein; the first photospecific binding protein is conjugated to an exosome-specific marker to form fusion protein I; and the second photospecific binding protein is conjugated to an NF-κB inhibitor to form fusion protein II.

Embodiment 14. The method of embodiment 13, in which fusion protein I and fusion protein II are reversibly bound via a first photospecific binding protein and a second photospecific binding protein.

Embodiment 15. The method of embodiment 13 or 14, wherein the first photospecific binding protein is conjugated to an exosome-specific marker so as to be positioned in a direction toward the inside of the exosome.

Embodiment 16. The method of any one of embodiments 13 to 15, wherein the first photospecific binding protein and the second photospecific binding protein are selected from the group consisting of CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, and PHR.

Embodiment 17. The method of embodiment 16, wherein the first photospecific binding protein is CIB or CIBN, and the second photospecific binding protein is CRY or PHR.

Embodiment 18. In any one of the preceding embodiments, the first photospecific binding protein is CRY or PHR, and the second photospecific binding protein is CIB or CIBN.

Embodiment 19. The method of embodiment 16, wherein the first photospecific binding protein is PhyB and the second photospecific binding protein is PIF.

Embodiment 20. The method of embodiment 16, wherein the first photospecific binding protein is PIF and the second photospecific binding protein is PhyB.

Embodiment 21. The method of embodiment 16, wherein the first photospecific binding protein is GIGANTEA and the second photospecific binding protein is FKF1.

Embodiment 22. The method of embodiment 16, wherein the first photospecific binding protein is FKF1 and the second photospecific binding protein is GIGANTEA.

Embodiment 23. The method of any one of embodiments 13 to 22, wherein the exosome-specific marker is selected from the group consisting of CD9, CD63, CD81, and CD82.

Embodiment 24. The method of any one of the preceding embodiments, wherein the exosomes have a diameter of about 50 nm to about 200 nm.

Embodiment 25. The method of any one of the preceding embodiments, wherein the exosomes have a diameter of about 50 nm to about 150 nm.

Embodiment 26. The method of any one of the preceding embodiments, wherein the composition is a pharmaceutical composition further comprising a physiologically acceptable carrier.

Embodiment 27. A composition comprising an exosome containing an NF-κB inhibitor as described in embodiments 1 to 16 for treating a disease induced by sepsis.

Embodiment 28. Use of a composition comprising an exosome containing an NF-κB inhibitor as described in embodiments 1 to 16 for treating a disease induced by sepsis.

Although the present disclosure has been described and illustrated herein with reference to various specific materials, processes and examples, it is understood that the disclosure is not limited to the specific combination of materials and processes selected for that purpose. Numerous variations of such details are implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered merely as exemplary, with the true scope and spirit of the present disclosure being set forth in the following claims. All references, patents and patent applications mentioned herein are incorporated herein by reference in their entirety.

Claims (12)

A composition for treating acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI) or sepsis , comprising an exosome containing super-repressor (SR)-IκB (srIκB) . The composition of claim 1 , wherein the composition is administered by a systemic route . The composition according to any one of claims 1 to 2 , wherein the composition is administered via oral, transdermal, intraperitoneal, intravenous, intramuscular, subcutaneous or mixed routes. The composition of claim 2 , wherein the systemic route is intraperitoneal or intravenous. The composition of any one of claims 1 to 4, further comprising an anti- inflammatory agent. The composition of any one of claims 1 to 5, wherein the exosome further comprises a photo-specific binding protein. The composition of any one of claims 1 to 6, wherein srIκB comprises an amino acid sequence having SEQ ID NO:1. the exosome comprising srIκB further comprises a first photospecific binding protein and/or a second photospecific binding protein;
a first photospecific binding protein is conjugated to an exosome-specific marker to form fusion protein I;
The composition of any one of claims 1 to 7, wherein a second photospecific binding protein is conjugated to srIκB to form fusion protein II. Use of exosomes containing super-repressor-IκB (srIκB) for the manufacture of a pharmaceutical composition for the treatment of acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI) or sepsis. The use according to claim 9, wherein the composition is administered by a systemic route. 10. The use according to claim 9, wherein the composition is administered via oral, transdermal, intraperitoneal, intravenous, intramuscular, subcutaneous or mixed routes. The use according to claim 10, wherein the systemic route is the intraperitoneal or intravenous route.