Small extracellular vesicles expressing a dominant negative AMPKα1 mutant for use in the treatment of obesity - Patent Application 20100223333

Abstract

The present invention relates to a population of small extracellular vesicles (sEVs) for use in the treatment of obesity in a subject in need thereof, the sEVs comprising at least one polynucleotide encoding a D168A dominant-negative AMP-activated protein kinase alpha 1 (AMPKalpha1-DN) mutant protein operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter, the sEVs being engineered to transiently express at least one fusion protein in their outer membrane comprising a neurotrophic rabies virus (RVG) peptide fused to a lysosome-associated membrane protein 2b. The population is highly safe and effective, since the sEVs can exert their effect in SF1-expressing neurons located in the ventromedial hypothalamus when administered presystemically.

Description

[Technical field]

[0001] The present invention relates to the field of nanobiomedicine. In particular, the present invention relates to the use of small extracellular vesicles administered via systemic routes for the treatment of obesity. [Background technology]

[0002] Obesity causes thousands of deaths annually worldwide, due to the many direct and indirect comorbidities associated with the condition, including cancer, cardiovascular disease and type 2 diabetes (T2D), but is the most preventable epidemic. 1-6 The most effective treatment for obesity is bariatric surgery, which not only reduces body weight but also improves T2D. However, the majority of obese subjects are not eligible for bariatric surgery. Moreover, in view of the harmful and dangerous side effects of bariatric surgery, there are increasing efforts to develop innovative anti-obesity drugs. 1、2、5 However, even though some current pharmacologically driven strategies show some beneficial results in weight loss, most of them exhibit undesirable side effects, mainly due to a lack of specificity.

[0003] Moreover, most of the current strategies are designed to target the food intake component of energy balance, but many of them do not trigger energy expenditure (EE). Indeed, since the regulation of body weight is complex and interconnected to multiple biological functions, it seems crucial to increase the specificity of treatments by identifying new molecular targets. 1、2、5 AMPK is a cellular gauge that is activated in conditions of low energy and promotes counterregulatory responses. 3、7~11 Recent evidence demonstrates that regulation of AMPK in the hypothalamus is a classical mechanism regulating energy balance. In particular, hormonal, pharmacological or genetic inhibition of AMPKα1 in the ventromedial hypothalamus (VMH) leads to increased sympathetic nervous system (SNS) activity that stimulates brown adipose tissue (BAT) thermogenesis, elevating EE and subsequently resulting in feeding-independent weight loss. 12~18At the cellular level, this action occurs in steroidogenic factor 1 (SF1) neurons of the VMH, and specific deletion of AMPKα1 promotes resistance to diet-induced obesity (DIO) and metabolic improvement in mice. 17、18 .

[0004] One of the keys to the effectiveness of anti-obesity drug development is its specificity of action. In this regard, the therapeutic potential of gene therapy is limited due to (i) its low stability in body fluids such as blood, which induces their rapid degradation after systemic injection, and (ii) its limited tissue-specific action. To counter these undesirable consequences after non-specific delivery, the past decades have provided some exciting breakthroughs through the expansion of nanomedicine strategies. Adenoviral or synthetic nanoparticle delivery strategies have been developed in regenerative medicine or for several pathologies. However, several problems, including repeated injections that induce inflammatory-undesirable responses, remain to be addressed by their use. It is therefore desirable to develop new biologically derived nanovesicles that can target tissues of interest and specifically deliver therapeutic genes, but without inducing immune responses or affecting other biological processes.

[0005] Extracellular vesicles (EVs) are a heterogeneous population of naturally occurring nano- to micro-sized membrane vesicles released by essentially all cell types. EVs have been shown to have therapeutic potential through target cell reprogramming, allowing for the regulation of cellular processes and secretions (molecules secreted by cells), and ultimately promoting tissue repair following target cell reprogramming. Summary of the Invention

[0006] The present invention provides the use of extracellular vesicles as systemically administered therapeutic vectors to deliver plasmids encoding dominant negative isoforms of AMPKα1 for use in the treatment or prevention of obesity. [Brief description of the drawings]

[0007] [Figure 1]FIG. 1. Generation and characterization of neuron-targeted dendritic cell-derived sEVs. (a) Example curves obtained by nanoparticle tracking analysis of samples of Lamp2b-RVG small extracellular vesicles (sEVs). The graph represents the concentration of sEVs (particles / mL) by size (nm). (b) Electron microscopy of generated Lamp2b-RVG sEVs shows a specific round shape and an average size of the vesicles of about 70 nm. (c) Western blotting using antibodies against ALIX, TSG101, CD9 and CD81 in Lamp2b-RVG sEVs. (d, e) Confocal micrographs of JAWS II cells treated for 2, 6 and 24 h with non-loaded sEVs (control), siRNA-Texas Red-loaded sEVs (sEV-siRNA Texas Red) or GFP plasmid-loaded sEVs (sEV-GFP). (f) Representative MAESTRO images of surviving mice after 30 min, 2, 4 and 6 h of intravenous injection of DID-labeled native (control) or DID-labeled Lamp2b-RVG sEVs (Lamp2b-RVG). (g) Ex vivo quantification of DID fluorescence in isolated organs (lungs, spleen, liver, heart and kidneys) 6 h after injection with DID-labeled native (control, n=3) or DID-labeled Lamp2b-RVG sEVs (Lamp2b-RVG, n=4). (h) Representative images of mouse brains, black circles demarcating the hypothalamus, taken 6 h after injection with DID-labeled native (control; n=3 mice / group) and DID-labeled Lamp2b-RVG sEVs (Lamp2b-RVG; n=4 mice / group). (i) Ex vivo quantification of DID fluorescence in mouse brains taken 6 hours after injection of DID-labeled native (control, n=3 mice / group) and DID-labeled Lamp2b-RVG sEVs (n=4 mice / group). (j) Representative pACCα western blot images of GT1-7 cells treated with control (unloaded) or SF1-AMPKα1-DN loaded sEVs for 24 hours (n=8 samples / group). β-actin was used as a control for protein loading. Black lines were inserted in the immunoblots when samples were loaded on the same gel but not side-by-side. (k) Quantification of pACCα in GT1-7 cells treated with control (unloaded, n=8 samples / group) or SF1-AMPKα1-DN loaded sEVs for 24 hours (n=8 samples / group). Data are presented as mean ± SEM.*P<0.05, **P<0.01 and ***P<0.001 versus control. Statistical significance was assessed by two-tailed Student's t-test. [Diagram 2] Effect of stereotaxic VMH injection of SF1-AMPKα1-DN loaded sEVs on energy balance. (a) Body weight change in grams for mice after stereotaxic VMH injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. Red arrow indicates start of injection (n=18 mice / group). (b) Daily food intake in grams for mice after stereotaxic VMH injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs (n=18 animals / group). (c) Representative pACCα and ACCα western blot images of VMH taken from mice 72 hours after stereotaxic VMH injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. β-actin was used as a control for protein loading. Black lines were inserted in immunoblots when samples were loaded on the same gel but not side-by-side. (d) Quantification of pACCα / ACCα expressed as % of control in the VMH, arcuate nucleus (ARC) and lateral hypothalamic area (LHA) (n=5–6 mice / group). (e,f) Representative thermography images (e) and BAT interscapular temperature quantification (average of 3 days) (f) of mice 72 h after stereotaxic VMH injection with control (unloaded, n=16 animals / group) or SF1-AMPKα1-DN loaded sEVs (n=17 animals / group). (g) Representative UCP1 western blot images of BAT harvested from mice 72 h after stereotaxic VMH injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. α-Tubulin was used as a control for protein loading. Black lines were inserted in immunoblots when samples were loaded on the same gel but not side-by-side. (h) Quantification of UCP1 protein expression in BAT harvested from mice 72 h after stereotaxic VMH injection with control (unloaded) or SF1-AMPKα1-DN-loaded sEVs (n=5–7 mice / group). Data are expressed as mean ± SEM. **P<0.01 and ***P<0.001 vs. control. Statistical significance was assessed by two-tailed Student's t-test. [Diagram 3]Figure 1. Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on hypothalamic AMPK activity. (a) In vivo expression of SF1-AMPKα1-DN plasmid at 24 hours in mice intravenously injected with SF1-AMPKα1-DN loaded sEVs. Representative agarose gel electrophoresis using specific SF1-AMPKα1-DN and HPRT primers. + Control is SF1-AMPKα1-DN plasmid. (b) Representative pACCα and ACCα Western blot images and quantification of pACCα in the VMH 72 hours after intravenous injection with control (unloaded, n=6 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=6 mice / group). β-actin was used as a control for protein loading. Black lines were inserted in immunoblots when samples were loaded on the same gel but not side-by-side. (c) Quantification of pACCα / ACCα expressed as % of control in the VMH. (d) Quantification of AMPK activity in the VMH 72 h after intravenous injection with control (unloaded, n = 6 mice / group) or SF1-AMPKα1-DN loaded sEVs (n = 5 mice / group). (e, f) Representative images showing Neurotrace500 / 525 (green), pACCα (magenta) positive cells and merged reactivity (e) as well as quantification of pACCα positive cell numbers (f) (quantification per field; 10–12 fields, 4 mice / group) in the VMH 24 h after intravenous injection with control (unloaded) sEVs or SF1-AMPKα1-DN loaded sEVs. Scale bars represent 20 μm. (g,h) Representative confocal images showing quantification of DAPI (blue), SF1 (red), pACCα (green) and merged reactivity (g) as well as pACCα fluorescence (h) in SF1 cells (quantification per field; 2–3 fields, 4 mice / group) in the VMH 24 h after intravenous injection with control (unloaded) or SF1-AMPKα1-DN-loaded sEVs (h). Arrows indicate pACC-positive SF1 cells. Scale bars represent 20 μm. (i,j) Time course of SF1-AMPKα1-DN plasmid expression in the VMH after a single intravenous injection.Representative agarose gel electrophoresis using specific SF1-AMPKα1-DN and HPRT primers (i) and quantification of SF1-AMPKα1-DN plasmid expression in the VMH (j) at different time points (n=4-5 mice / group). MWM, molecular weight marker. Data are expressed as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. control. Statistical significance was assessed by two-tailed Student's t-test. [Figure 4]FIG. 1. Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on energy balance. (a, b) Body weight change in grams (a) and percentage (b) of mice after intravenous injection of control (unloaded) or SF1-AMPKα1-DN loaded sEVs every 3 days for 6 days. Red arrows indicate injections (n=24-25 mice / group). (c) Food intake in grams of mice after intravenous injection of control (unloaded) or SF1-AMPKα1-DN loaded sEVs every 3 days for 6 days. Red arrows indicate injections (n=25 mice / group). (d) Daily food intake in grams of mice after intravenous injection of control (unloaded) or SF1-AMPKα1-DN loaded sEVs every 3 days for 6 days (n=25 mice / group). (e-g) Energy expenditure (EE, e), respiratory quotient (RQ, f) and locomotor activity (LA, g) during the light and dark phases in mice after intravenous VMH injection of control (unloaded, n = 6 mice / group) or SF1-AMPKα1-DN loaded sEVs (n = 5 mice / group) every 3 days for 6 days. (h) Representative NMR images showing adipose tissue (AT) on fat (AT on) and fat-free (AT off), total AT, subcutaneous AT (scAT) and visceral AT (VAT) images obtained by subtracting AT on by AT off in mice after intravenous VMH injection of control (unloaded, n = 7 mice / group) or SF1-AMPKα1-DN loaded sEVs (n = 8 mice / group) every 3 days for 6 days. (i-k) Quantification of AT mass in grams; total AT mass (i), scAT (j) and vAT (k) of mice after intravenous VMH injection of control (unloaded, n=7 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=8 mice / group) every 3 days for 6 days. (l,n) Body weight (l) and body weight change in grams (m) and percentage (day 28-d1) (n) of mice after intravenous injection of control (unloaded) or SF1-AMPKα1-DN loaded sEVs every 3 days for 28 days (n=15 mice / group) followed by a 14-day washout (n=8 mice / group). Red arrows indicate injections.(o,p) Food intake (o) and daily food intake (p) of mice after intravenous injection of control (non-loaded) or SF1-AMPKα1-DN loaded sEVs every 3 days for 28 days followed by a 14-day washout (n=15 mice / group). Red arrows indicate injections. (q-s) Body weight change in grams during the entire treatment period (q) and during each treatment week (r) of mice after intravenous injection of control (non-loaded) or SF1-AMPKα1-DN loaded sEVs every 3 days for 6 days, followed by a 2-week washout and injection again every 3 days for 6 days. Food intake in grams (s) of mice after intravenous injection of control (non-loaded, n=7 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=7 mice / group) following the same protocol. Red arrows indicate injections. Data are presented as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 versus control. Statistical significance was assessed by two-tailed Student's t-test. [Diagram 5]Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on BAT thermogenesis. (a-c) Representative BAT thermography images (a), daily BAT temperatures (b) and mean BAT temperature quantification (c) of mice injected into the tail vein every 3 days for 6 days with control (unloaded) or SF1-AMPKα1-DN loaded sEVs (n=10 mice / group) [n=69-70 mice / group; box plots show median (middle line), 25th, 75th percentiles (boxes) and 10th-90th percentiles (whiskers; minimum and maximum, respectively)]. Red arrows indicate injections. (d,e) Correlation analysis of BAT temperature (°C) (d) and food intake (e) with body weight change expressed in grams (n=60 individuals / group) of mice injected into the tail vein every 3 days for 6 days with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. (f-h) Representative BAT thermography images (f), daily BAT temperature time course (g) and daily BAT temperature histograms (h) on days 4, 14 and 23 of mice after intravenous injection of control (unloaded, n=8 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=8 mice / group) every 3 days for 6 days, followed by a 2-week washout and injection again every 3 days for 6 days. (i,j) Representative tail base thermography images (i) and mean tail base temperature quantification (j) of mice after intravenous injection of control (unloaded, n=8 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=8 mice / group) every 3 days for 6 days, followed by a 2-week washout and injection again every 3 days for 6 days. Data are expressed as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. control. Statistical significance was assessed by two-tailed Student's t-test, except for BAT temperatures on days 2 and 23 in the crossover experiment (g), which used a one-tailed Student's t-test. [Figure 6]Figure 1. Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on BAT thermogenic molecular pathways. (a, b) Representative UCP1, UCP3, PGC1α and PGC1β western blot images (a) and quantification of their expression (b) in BAT of mice after intravenous injection of control (unloaded, n=8-16 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=10-18 mice / group) every 3 days for 6 days. α-Tubulin was used as a control for protein loading. Black lines were inserted in the immunoblots when samples were loaded on the same gel but not side-by-side. (c, d) Representative axial, sagittal and coronal PET-CT scan images (c) and standardized uptake value (SUV) BAT / liver ratios (d) showing basal levels and BAT after injection of control (unloaded, n=5 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=5 mice / group). (e, f) Representative scWAT immunohistochemistry with anti-UCP1 antibody showing UCP1 staining (e) and quantification (f) of UCP1 stained areas in scWAT from mice after injection of control (unloaded, n=7 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=7 mice / group). Scale bar represents 100 μm. Data are presented as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. control. Statistical significance was assessed by two-tailed Student's t-test. [Figure 7]Effect of adrenergic blockade on systemic treatment with SF1-AMPKα1-DN loaded sEVs. (a, b) Representative BAT SNA traces (a) and their quantification (b) (total, efferent and afferent signals) in spikes / sec of DIO mice injected with control (unloaded; n=6 mice / group) or SF1-AMPKα1-DN sEVs (n=6 mice / group) via the tail vein for 24 h. (c-f) Body weight change (grams, c), daily food intake (grams, d), representative BAT thermography images (e) and mean BAT temperature quantification (f) of mice injected with control (unloaded; n=6 mice / group), SF1-AMPKα1-DN loaded sEVs alone (n=6 mice / group) or in the presence of the specific β3-AR antagonist, SR59230A (n=6 mice / group). (g,h) Representative UCP1 western blot images (g) and quantification of its expression (h) in BAT of mice after intravenous injection of control (unloaded; n=6 mice / group), SF1-AMPKα1-DN loaded sEVs alone or in the presence of a specific β3-AR antagonist, SR59230A (n=6 mice / group). α-Tubulin was used as a control for protein loading. Black lines were inserted in immunoblots when samples were loaded on the same gel but not side-by-side. Data are expressed as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. control. #P<0.05 and ##P<0.01 SF1-AMPKα1-DN vs. SF1-AMPKα1-DN+SR59230A. Statistical significance was assessed by two-tailed ANOVA. [Figure 8]Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on energy balance in thermoneutral and UCP1 knockout mice. (a, b) Body weight change in grams (a) and percentage (b) of mice after intravenous injection of control (unloaded, n=9 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=9 mice / group) every 3 days for 6 days under thermoneutral conditions (30° C.). (c, d) Food intake (c) and daily food intake (d) of mice after intravenous injection of control (unloaded, n=9 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=9 mice / group) every 3 days for 6 days under thermoneutral conditions (30° C.). (e,f) Representative BAT thermography images (e) and BAT temperature interscapular temperature quantification (f) of mice after intravenous injection every 3 days for 6 days with control (unloaded, n=9 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=9 mice / group) placed under thermoneutral conditions. (g,h) Representative UCP1 Western blot images (g) and quantification of UCP1 expression (h) in BAT of mice after intravenous injection every 3 days for 6 days with control (unloaded, n=5 mice / group) or SF1-AMPKα1-DN loaded sEVs (n=7 mice / group). α-Tubulin was used as a control for protein loading. Black lines were inserted in immunoblots when samples were loaded on the same gel but not side-by-side. (i,j) Body weight change in grams of wild-type (ucp1+ / +, i, n=7 mice / group) and ucp1 null mice (ucp1- / -, j, n=10 mice / group) after a single intravenous injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. Red arrows indicate injections. (k,l) Daily food intake of wild-type (k, n=7 mice / group) and ucp1 null mice (l, n=10 mice / group) after a single intravenous injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. (m-p) Representative BAT thermography images (m,o) and BAT interscapular temperature quantification (n,p) of wild-type (m,n,n=7 mice / group) and ucp1 null mice (o,p,n=10 mice / group) after a single intravenous injection with control (unloaded) or SF1-AMPKα1-DN-loaded sEVs. Data are presented as mean ± SEM.*P<0.05, **P<0.01 and ***P<0.001 versus control. Statistical significance was assessed by two-tailed Student's t-test. [Figure 9] Characterization of SF1-AMPKα1-DN loaded neuron-targeted dendritic cell-derived sEVs. (a) Western blotting using an antibody against Lamp2b in native and Lamp2b-RVG sEVs. (b) Quantification of Lamp2b levels in native (n=4 samples / group) and Lamp2b-RVG (n=5 samples / group) sEVs in % native control. (c) Western blotting with an antibody against GRP94 in Jaws II cells (lane 1), unmodified native (lane 2) and Lamp2b-RVG sEVs (lane 3). (d) Circular representation of the plasmid encoding SF1-AMPKα1-DN. (e) Example of curves obtained by nanoparticle tracking analysis of samples of native (left panel) and SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs (right panel). Graphs represent concentration of sEVs (particles / mL) according to size (nm). (f) Electron microscopy of SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs showing the characteristic round shape and average size of the vesicles of about 70 nm. (g) Agarose gel electrophoresis of AMPK and GAPDH native (lane 1), Lamp2b-RVG (lane 2), SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs (lane 3) and negative control H2O (lane 4). (h) Agarose gel electrophoresis of SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs treated with DNAse (lane 1), DNAse+TritonX-100 0.2% (lane 2) and TritonX-100 0.2% (lane 3) AMPK and GAPDH. (i) Quantification of pACCα / ACCα expressed in % of control in primary astrocytes treated with native (n=6 samples / group) and Lamp2b-RVG (n=6 samples / group) sEVs for 24 h. (j) Quantification of pACCα / ACCα expressed in % of control in Neuro2A cells treated with native (n=6) and Lamp2b-RVG (n=6) sEVs for 24 h. Data are presented as mean ± SEM. ***P<0.001 vs. control. Statistical significance was assessed by two-tailed Student's t-test. [Figure 10] Control of hypothalamic nuclei dissections. Quantification of Pomc, Sf1 and Hcrt / orexin mRNA levels in ARC, VMH and LHA dissections [n=19-20 mice / group; box plots show median (midline), 25th, 75th percentiles (boxes) and 10th-90th percentiles (whiskers; minimum and maximum, respectively)]. Data are expressed as mean ± SEM. ***P<0.001 vs. Pomc ARC, Sf1 VMH and Hcrt LHA. Statistical significance was assessed by two-tailed Student's t-test. [Figure 11] FIG. 1. Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on hypothalamic AMPK activity. (a) Representative confocal images showing GFAP (green), pACCα (magenta) and merged reactivity in brain sections 24 hours after intravenous injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. Scale bar represents 20 μm. (b) Representative confocal images showing Iba1 (green), pACCα (magenta) and merged reactivity in brain sections 24 hours after intravenous injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. Scale bar represents 20 μm. (c) Negative control for pACCα and SF1 double immunofluorescence. Representative confocal images showing DAPI (blue) with or without SF1 (red), Alexa594 with or without pACCα (green), Alexa488 with or without pACCα (green) and merged reactivity in brain sections. Scale bar represents 20 μm. (d) Quantification of pACCα fluorescence in the ARC, DMH and PVH (quantification per field; 4–12 fields) 24 h after intravenous injection with control (unloaded) or SF1-AMPKα1-DN-loaded sEVs. Data are presented as mean ± SEM. Statistical significance was assessed by two-tailed Student's t-test. [Figure 12]Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on central and peripheral tissues. (a) Quantification of pACCα / ACCα levels in the cortex, thalamus and cerebellum 28 days after intravenous injection with control (unloaded, n=6-7 mice / group) or SF1-AMPKα1-DN loaded (n=7 mice / group) sEVs, expressed as % of control. (b) Quantification of pACCα / ACCα levels in the liver, adrenal gland, testis, BAT, heart and skeletal muscle 28 days after intravenous injection with control (unloaded, n=6-7 mice / group) or SF1-AMPKα1-DN loaded (n=7 mice / group) sEVs, expressed as % of control. (c) Quantification of pACCα / ACCα levels in brown adipocytes after 24 h incubation with control (unloaded, n=5 samples / group) or SF1-AMPKα1-DN loaded (n=5 samples / group) sEVs, expressed as % of control. (d) Quantification of circulating testosterone levels expressed in ng / ml 28 days after intravenous injection with control (unloaded, n=7 mice / group) or SF1-AMPKα1-DN loaded (n=7 mice / group) sEVs. (e) Quantification of mRNA levels of steroidogenic enzymes (STAR, p450scc and 17β-HSD3) in testes, expressed as % of control, 28 days after intravenous injection with control (unloaded, n=7 mice / group) or SF1-AMPKα1-DN loaded (n=7 mice / group) sEVs. (f) Quantification of circulating CORT levels expressed in ng / ml 28 days after intravenous injection with control (unloaded, n=7 mice / group) or SF1-AMPKα1-DN loaded (n=6 mice / group) sEVs. (g) Quantification of mRNA levels of steroidogenic enzymes (STAR ​​and p450scc) in the adrenal gland expressed as % of control 28 days after intravenous injection with control (unloaded, n=5 mice / group) or SF1-AMPKα1-DN loaded (n=6 mice / group) sEVs. (h) Quantification of circulating LH levels expressed in ng / ml 28 days after intravenous injection with control (unloaded, n=6 mice / group) or SF1-AMPKα1-DN loaded (n=6 mice / group) sEVs.(i) Quantification of LHβ subunit mRNA levels in the pituitary gland after 28 days of intravenous injection with control (unloaded, n=6 mice / group) or SF1-AMPKα1-DN-loaded (n=7 mice / group) sEVs, expressed as % of control. (j) Quantification of BAT UCP1 levels 1, 2, 3 and 7 days after a single tail vein injection of control (unloaded, n=5–6 mice / group) or SF1-AMPKα1-DN (n=4–5 mice / group) sEVs on day 0. Values ​​are expressed as % of control. Data are expressed as mean ± SEM. *P<0.05 and **P<0.01 vs. control. Statistical significance was assessed by two-tailed Student's t-test. [Figure 13] FIG. 1. Effect of systemic treatment with SF1-AMPKα1-DN sEVs on circulatory and hemodynamic parameters. (a-i) Quantification of circulating leptin (a), GDF15 (b), IL-6 (c), IP-10 (d), triglycerides (e), cholesterol (f), NEFA (g), AST (h) and ALT (i) after 28 days of intravenous injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs (n=5-13 mice / group). (jm) Quantification of heart rate (j, n=6 mice / group), systolic arterial pressure (k, n=6 mice / group), diastolic arterial pressure (l, n=6 mice / group) and mean arterial pressure (m, n=6 mice / group) 24 hours after intravenous injection with control (unloaded) or SF1-AMPKα1-DN loaded sEVs. Data are expressed as mean ± SEM. Statistical significance was assessed by two-tailed Student's t test. [Figure 14]Effect of systemic treatment with SF1-AMPKα1-DN sEVs on BAT and skeletal muscle thermogenic markers. (a) Quantification of UCP1 protein levels in BAT 28 days after intravenous injection with control (unloaded, n=7 mice / group) or SF1-AMPKα1-DN loaded (n=7 mice / group) sEVs. (b) Quantification of mRNA levels of thermogenic markers (Ucp3, Gpd2, PParγ, Sln, Ryr1, Atp2a2) in skeletal muscle 28 days after intravenous injection with control (unloaded, n=6–7 mice / group) or SF1-AMPKα1-DN loaded (n=6–7 mice / group) sEVs expressed as % of control. Data are presented as mean ± SEM. *, **P<0.05 vs. control. Statistical significance was assessed by Student's t-test. [Figure 15] FIG. 1. Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on energy balance in db / db mice. (A-B) Body weight; (C-D) Body weight change; (E-H) Daily and cumulative food intake; (I and K) Fat mass and (J and L) Serum triglyceride levels of wild-type and db / db mice treated intravenously with control (unloaded; n=8 wild-type mice; n=8 db / db mice) or SF1-AMPKα1-DN loaded (n=7 wild-type; n=7 db / db mice) sEVs. Data are expressed as MEAN±SEM. Statistical significance was determined by mixed effects analysis (A-D) or two-tailed Student's t-test (E-L). *P<0.05 vs. control. [Figure 16]Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on BAT thermogenesis and WAT browning in db / db mice. (A-B) Representative pACCα and ACCα Western blot images and levels in the VMH (n=7 mice / group); (C-D) Representative BAT thermography images and BAT temperature (day 14; n=7-8 mice / group); (E-F) Representative UCP1 Western blot images and levels in BAT (n=6 mice / group); (G-H) Representative BAT UCP1 staining and levels (n=7-8 mice / group) and (I-J) Representative sWAT UCP1 staining and levels (n=7-8 mice / group) of wild-type and db / db mice intravenously injected with control or SF1-AMPKα1-DN loaded sEVs. In Western blot analyses (A–B and E–F), β-actin (in VMH; not shown) and α-tubulin (in BAT) were used as controls for protein loading. Black lines were inserted in immunoblots when samples were loaded on the same gel but not side-by-side. Data are expressed as MEAN ± SEM. Statistical significance was determined by two-tailed Student's t test, except for temperature of BAT in db / db mice (D), where one-tailed Student's t test was used. *P<0.05, **P<0.01 vs. control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Invention The present invention provides a population of small extracellular vesicles (sEVs) comprising at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKα1-DN) mutant protein, the amino acid sequence of which consists of SEQ ID NO: 1, 39 or 41, wherein the AMPKα1-DN mutant protein is operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter having at least 98%, preferably 100%, sequence identity to SEQ ID NO: 3, and the sEVs are engineered to express at least one fusion protein in their outer membrane comprising a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b.

[0009] Preferably, the fusion protein comprising a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises SEQ ID NO:5, or a sequence having at least 90% sequence identity to SEQ ID NO:5.

[0010] Preferably, the population of sEVs is for use in the treatment or prevention of obesity, such as diet-induced obesity and / or genetic obesity. Preferably, the genetic obesity is leptin receptor (LEPR) deficiency induced obesity.

[0011] Preferably, the population of sEVs is for use in ameliorating or reducing the rebound effect following washout of an obesity treatment, and preferably, said amelioration or reduction in the rebound effect is measured at least 5 days following washout of the treatment.

[0012] Preferably, administration of the small extracellular vesicles is systemic, preferably intravenous.

[0013] The present invention further provides a population of small extracellular vesicles (sEVs) that, when administered systemically, are capable of significantly reducing activation levels of AMP-activated protein kinase (AMPK) in SF1-expressing neurons located in the ventromedial hypothalamus (VMH) compared to the lack of effect on AMPK activity in untreated SF1-expressing cells, said reduction not being significantly reduced in other SF1-expressing tissues, e.g. non-neuronal tissues / organs, preferably selected from the list consisting of the adrenal gland, testis or pituitary gland, and wherein said population of small extracellular vesicles (sEVs) comprises at least one sEV encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKα1-DN) mutant protein. The population comprises one polynucleotide, wherein the amino acid sequence of the AMPKα1-DN mutant protein consists of SEQ ID NO:1, 39 or 41, the AMPKα1-DN mutant protein is operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter having at least 98%, preferably 100%, sequence identity to SEQ ID NO:3, the sEVs are engineered to express at least one fusion protein in their outer membrane comprising a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and the population is for use in the treatment of obesity in a subject in need thereof, preferably via a systemic administration route.

[0014] Preferably, the nucleotide sequence of the AMPKα1-DN mutant protein consists of SEQ ID NO: 2. Preferably, the fusion protein comprising a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises SEQ ID NO: 5, or a sequence having at least 90% sequence identity to SEQ ID NO: 5.

[0015] Preferably, the use is the amelioration or reduction of the rebound effect following washout of a treatment for obesity, said amelioration or reduction of the rebound effect being measured at least 5 days after washout of the treatment. Preferably, the treatment comprises reversing or ameliorating obesity.

[0016] Preferably, the small extracellular vesicles have a size distribution of 30-150 nm. Preferably, the sEVs are characterized by lacking the GRP94 marker. Preferably, the sEVs are produced or obtained from immature antigen-presenting cells characterized by having a statistically significant decrease in expression of at least one T cell activator molecule compared to the expression of the T cell activator molecule in mature antigen-presenting cells. Preferably, the antigen-presenting cells are dendritic cells, preferably a JAWSII cell line, and the at least one T cell activator molecule is one or more T cell activator molecules selected from the group consisting of major histocompatibility complex II (MHC-II), cluster of differentiation 80 (CD80) or cluster of differentiation 86 (CD86), or a combination thereof.

[0017] Preferably, the sEVs are exosomes.

[0018] General definition It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element of the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0019] The term "about" with respect to a given amount or content is meant to include a deviation of ±10 percent.

[0020] As used herein, the conjunctive term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning and thus meet the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more options is also understood to be within the meaning and thus meet the requirements of the term "and / or".

[0021] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are understood to mean the inclusion of a recited integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced with the terms "containing" or "including", or, as sometimes used herein, with the term "having". Any of the foregoing terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the invention, may be replaced, but is less preferred, with the term "consisting of".

[0022] As used herein, "consisting of" excludes elements, steps, or ingredients not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0023] As used herein, the term "extracellular vesicles" (EVs) refers to lipid bilayer-bound vesicles released from living cells into the extracellular environment. These vesicles lack a functional nucleus and are unable to replicate. Traditionally, EVs can be broadly divided into two main subtypes with respect to their characteristics and biogenesis pathways: ectosomes and small extracellular vesicles (sEVs). Ectosomes have a size distribution of 100 nm to 1000 nm and are generated by cytoplasmic membrane budding. sEVs, including exosomes, are smaller in diameter (usually 30 to 150 nm) and are released by fusion of multivesicular bodies (MVBs) with the plasma membrane. The components of sEVs indicate their cellular origin and potential biological functions. In the context of the present invention, sEVs or exosomes are preferably produced in antigen-presenting cells, as described in detail below.

[0024] As used herein, the phrase "subject in need thereof" includes a subject, e.g., a mammalian subject, preferably a human, who would benefit from administration of a population of sEVs disclosed herein. A subject in need thereof may be a person who is trying to lose weight or is in need of weight loss. The subject may be a female, preferably a human female. Preferably, the subject is a male, more preferably a human male. More preferably, the subject is obese or in need of treatment for obesity. The terms "individual", "patient" or "subject" are used interchangeably in this application and are not meant to be limiting in any way. An "individual", "patient" or "subject" may be of any age, sex and physical condition.

[0025] "Nucleic acid," "nucleic acid molecule," "oligonucleotide," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single-stranded form or as a double-stranded helix.

[0026] As used herein, the term "gene" or "coding sequence" refers to an in vitro or in vivo polynucleotide sequence that encodes a gene product. In some instances, a gene consists or consists essentially of a coding sequence, i.e., a sequence that encodes a gene product.

[0027] As used herein, the term "AMP-activated protein kinase" (AMPK) refers to an enzyme that plays a role in cellular energy homeostasis, activating glucose and fatty acid uptake and oxidation primarily when cellular energy is low. It belongs to a highly conserved eukaryotic protein family. It consists of three proteins (subunits) that together make a functional enzyme conserved from yeast to humans. It is expressed in many tissues, including the liver, brain, and skeletal muscle. In response to the binding of AMP and ADP, the net effect of AMPK activation is stimulation of hepatic fatty acid oxidation, ketogenesis, stimulation of skeletal muscle fatty acid oxidation and glucose uptake, inhibition of cholesterol synthesis, fatty acid synthesis and triglyceride synthesis, inhibition of adipocyte lipogenesis, and regulation of insulin secretion by pancreatic beta cells. AMPK is a heterotrimeric protein complex formed by α, β, and γ subunits. As used herein, "AMPKα" refers to the subunit alpha (α) of the AMPK protein. Due to the existence of its component isoforms, there are 12 types of AMPK in mammals, each of which may have different tissue localizations and different functions under different conditions. The α, β and γ subunits can also be found in different isoforms: the γ subunit can exist as either γ1, γ2 or γ3 isoforms, the β subunit can exist as either β1 or β2 isoforms, and the α subunit can exist as either α1 or α2 isoforms. As used herein, "AMPKα" refers to isoform 1 of the subunit α of the AMPK protein. As used herein, the term "dominant negative AMPKα1 mutant protein" (AMPKα1-DN) refers to an inactive isoform of AMPKα1, preferably rat AMPKα1 protein, by modification of its active site with at least the point mutation D168A, amino acid position (168) expressed with respect to the wild-type rat AMPKa1 sequence, preferably the wild-type rat AMPKa1 sequence of SEQ ID NO: 40.In the context of the present invention, "AMPKα1-DN" refers to an inactive isoform of AMPKα1, preferably a rat AMPKα1 protein, by modifying its active site with at least the point mutation D168 relative to the wild-type rat AMPKa1 sequence, preferably the wild-type rat AMPKa1 sequence of SEQ ID NO: 40, and the sequence of the AMPKα1-DN protein may be the sequence of the rat AMPKα1 protein (as shown in the examples), or the human homologue of the AMPKα1 protein, or a homologue sequence of the rat AMPKα1 protein corresponding to the species to which the treatment is applied, such as a humanized or codon-optimized AMPKα1 protein for treatment in humans. Alternatively, rat sequences of AMPKα1 protein can be used to treat other species, such as humans, as rat AMPKα1-DN has been shown to be effective in other animals, such as mice (see Seoane-Collazo et al, 2018, SF1-Specific AMPKα1 Deletion Protects Against Diet-Induced Obesity. Diabetes. 2018 Nov;67(11):2213-2226) and human cells (Stein et al. 2000. The regulation of AMP-activated protein kinase by phosphorylation. Biochem. J. (2000) 345,437-443). When using AMPK human sequences, the sequences can also or alternatively contain other point mutations, such as the T172A mutation.

[0028] As used herein, the term "nuclear receptor steroidogenic factor-1 (SF-1)" refers to a transcription factor essential for the terminal differentiation and maintenance of the ventromedial nucleus neuronal population.

[0029] As used herein, the term "SF1-expressing cell or tissue" refers to a cell or tissue capable of expressing the SF1 transcription factor. Preferably, the SF1-expressing cell is a SF1-expressing neuron located in the VMH region.

[0030] The hypothalamus is organized into anatomically distinct interconnected hypothalamic nuclei, including the arcuate nucleus (ARC), the ventromedial nucleus (VMH), the paraventricular nucleus (PVH), the lateral hypothalamus (LHA) and the dorsomedial nucleus (DMH), which are involved in regulating energy metabolism, among others.Therefore, as used herein, the term "ventromedial hypothalamic nucleus" (VMH) refers to the hypothalamic region.The VMH region is known to be involved in feeding behavior and energy expenditure regulation through brown adipose tissue (BAT) thermogenesis.The VMH is associated with other nuclei through axonal projections.

[0031] As used herein, "pharmacologically acceptable carrier" or "pharmacologically acceptable diluent" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents compatible with pharmaceutical administration. The use of such media and agents for pharma- ceutical active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include, without limiting the scope of the invention, the following: additional buffering agents; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; lactitol, stachysulfate, glycerol ... organic sugars or sugar alcohols such as ose, mannose, sorbose, xylose, ribose, ribitol, myo-initose, myo-inititol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone.

[0032] The amino acid residue positions or sites of a protein or polypeptide sequence are preferably numbered sequentially starting from the first amino acid residue, which is then located at position 1. For example, a 137 amino acid protein has residues numbered from 1 (the first amino acid residue) up to 137 (the last amino acid residue). Preferably, the first position or position number 1 corresponds to the first amino acid residue located at the five prime (5') end of the polypeptide chain, which has a nitrogen atom or a free amino group. Thus, the numbering preferably starts from the first amino acid residue at the N-terminus or 5'-terminus of the protein or polypeptide and ends at the 3'-terminus or C-terminus of the protein or polypeptide. Preferably, the amino acid residue positions are numbered using the amino acid sequence of the translated mature protein.

[0033] The term "sequence identity" or "percent identity" in the context of two or more nucleotide, polypeptide or protein sequences refers to two or more sequences or subsequences that are the same ("identical") or have a specified percentage of identical nucleotides or amino acid residues ("percent identity") when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm, preferably the BLAST alignment tool, or alternatively by visual inspection. "Sequence identity" or "percent identity" can be determined by calculating the number of identical nucleotides or amino acids at the same positions in a nucleic acid, polypeptide or protein. Calculating percent identity includes determining the optimal alignment between two or more sequences. Alignment can take into account insertions and deletions (i.e., "gaps") in each of the sequences tested, including, but not limited to, non-coding regions of nucleic acids and truncations or extensions of polypeptide sequences. Percent identity can be determined using computer programs and algorithms such as the Basic Local Alignment Search Tool (BLAST). BLAST is one of many resources provided by the National Center for Biotechnology Information. Since genetic code is degenerate and more than one codon can code for a given amino acid, the coding region of a nucleic acid is considered to be identical if the nucleic acid codes for the same polypeptide.Therefore, percent identity can also be calculated based on the polypeptide coded by the nucleic acid.Percent identity can be calculated based on the full-length consensus genome sequence, or based on a portion of the genome sequence, for example, but not limited to, based on individual open reading frames (ORFs).

[0034] "Percent (%) amino acid sequence identity" with respect to a protein or polypeptide described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference sequence (i.e., the protein or polypeptide from which it is derived) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, using publicly available computer software such as BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

[0035] Preferably, "percent identity" as used herein is determined in the context of local alignment, i.e., based on the alignment of the area of ​​local similarity between nucleobase sequences, as opposed to the global alignment that aims to align two sequences over their entire span.Therefore, in the context of the present invention, percent identity is preferably calculated based only on local alignment comparison algorithm.

[0036] explanation The aim of this study is to develop a new strategy to treat or prevent obesity by specifically reducing the activity of hypothalamic AMPK in SF1 neurons. For this purpose, we used small extracellular vesicles (sEVs) as carriers of a plasmid encoding a dominant-negative AMPKα1 mutant (AMPKα1-DN). sEVs originate from multivesicular bodies and contain proteins, lipids, and genetic information that can modify the phenotype and function of target cells, allowing them to play a key role in physiology and pathophysiology. To confer specificity to the expression of this AMPKα1-DN mutant only in hypothalamic SF1-expressing neurons, the SF1 promoter was used to drive its expression. Notably, to avoid any invasive cranial surgery / procedures, the aim of this study was also to specifically target AMPK in hypothalamic SF1 neurons using a systemic administration route, making them accessible for potential therapeutic use.

[0037] Thus, the present invention provides SF1-AMPKα1-DN loaded sEVs that are systemically administered for use in the treatment or prevention of obesity. Large amounts of sEVs were generated using immature dendritic cells. To confer neural targeting capability to the produced sEVs, immature dendritic cells were genetically modified to transiently express a fusion protein consisting of (i) lysosomal associated membrane protein 2b (Lamp2b), a protein highly expressed in the sEV membrane, fused to (ii) a specific glycoprotein derived from the neurotrophic rabies virus (RVG) that allows crossing of the blood-brain barrier (BBB) ​​via binding to the nicotinic acetylcholine receptor (nAChR).

[0038] To test the efficacy of this strategy, the phosphorylation levels of acetyl-CoA carboxylase α (pACCα), a regulatory kinase that is the primary target of the enzyme AMPK, were measured in obese mice treated with SF1-AMPKα1-DN-loaded Lamp2b-RVG sEVs via central and systemic administration routes. The results demonstrated that SF1-AMPKα1-DN sEVs induced feeding-independent weight loss. The levels of pACCα, used to control the degree of AMPK inhibition in hypothalamic extracts, were also specifically reduced in the VMH region, but not in the ARC or LHA regions, which was associated with increased BAT thermogenesis (Figure 2). Upon systemic administration, the transgene was only detected in VMH samples, but not in any of the other evaluated organs, including those expressing SF1 (i.e., adrenal gland, pituitary gland, and testis) or those not expressing SF1 (i.e., BAT, liver, skeletal muscle, and heart) (Figure 3a), indicating that the effect of this treatment was restricted to neural cells, particularly SF1-expressing neurons in the VMH, demonstrating that this strategy is highly specific. Furthermore, SF1-AMPKα1-DN-loaded sEVs induced a significant decrease in the levels of ACCα phosphorylation and AMPK activity in the VMH (Figure 3).

[0039] Furthermore, we investigated whether treatment with sEVs in obese mice would have a rebound effect, i.e., a rapid recovery of weight loss to catch up with the weight of control mice. Figure 4 shows that the effect of sEVs persisted when treatment was stopped. Mice treated with SF1-AMPKα1-DN loaded sEVs did not show weight recovery until 2 weeks after injections were stopped (washout) when compared to mice treated with control sEVs. This indicates that the weight loss is not transient and the washout period does not imply a rebound effect in treated mice.

[0040] Taken together, these data support that SF1-AMPKα1-DN mutant-loaded Lamp2b-RVG sEVs are suitable to specifically downregulate or inhibit AMPK activity in specific SF1-expressing neurons to induce weight loss, and thus sEVs represent a potential therapeutic strategy for treating or preventing obesity.

[0041] In a first aspect, the present invention relates to the systemic administration of a population of small extracellular vesicles (sEVs) for use in the treatment or prevention of obesity in a subject in need thereof. In particular, in a first aspect, the present invention relates to the systemic administration of a population of sEVs for use in the treatment or prevention of obesity in a subject in need thereof, the sEVs comprising at least one polynucleotide encoding an AMPK protein, preferably an AMPKα, preferably an AMPKα1, most preferably an AMPKα1-DN (dominant negative AMP-activated protein kinase α1 mutant) protein, operably linked and under the control of a steroidogenic factor 1 (SF1) promoter, the sEVs being engineered to preferably transiently express in their outer membrane at least one fusion protein comprising a neurotrophic rabies virus (RVG) peptide fused to lysosomal associated membrane protein 2b (Lamp2b). A coding sequence and a gene expression control sequence or promoter are said to be operably linked when they are linked in such a way that the expression or transcription and / or translation of the coding sequence is under the influence or control of the gene expression control sequence. For example, a dominant negative AMPKα1 mutant protein (AMPKα1-DN) is operably linked to the SF1 promoter such that the expression level of AMPKα1-DN is regulated by the SF1 promoter. In one embodiment, the obesity is diet-induced obesity and / or genetic obesity. In one embodiment, the obesity is genetic obesity, preferably leptin receptor (LEPR) deficiency-induced obesity.

[0042] Preferably, the population of sEVs is exosomes. In one embodiment, the population of sEVs or exosomes is a substantially pure population. Preferably, the sEVs or exosomes are isolated. In the context of the present invention, the term "isolated" indicates that the sEVs or exosomes or the population thereof are not in the environment or cell culture in which they were produced. The sEVs or exosomes or the population thereof are substantially separated from the surrounding environment or cell culture. In some embodiments, the population is substantially pure or enriched in sEVs or exosomes, comprising at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of the sEVs or exosomes. In one embodiment, the population is pure in sEVs or exosomes. They are released by many cell types as a means of communicating with other cells, and therefore cellular contents may be removed from the population. The cargo of extracellular vesicles includes proteins, lipids, nucleic acids, and membrane receptors of the cells from which they are derived. The term isolated also encompasses populations of sEVs or exosomes that have been removed from the environment or cell culture, for example from the supernatant or conditioned medium from which they are derived.

[0043] In one embodiment, the sEVs or exosomes are spherical or round. Furthermore, the sEVs or exosomes may have a size greater than 2 nm. The sEVs or exosomes may have a size greater than 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm or 160 nm. The exosomes may have a size substantially greater than 160 nm. The sEVs or exosomes may have a size distribution in the range of 30 nm to 50 nm, 30 nm to 100 nm, preferably 30 nm to 150 nm or 30 nm to 200 nm, etc. The size distribution may be determined by various means. In principle, the size may be determined by size fractionation and filtration through membranes with associated size cutoffs. The size of the sEVs or exosomes can then be determined by following the separation of the component proteins on SDS-PAGE or by biological assays. Size can also be determined by electron microscopy or nanoparticle tracking analysis (NTA).

[0044] The population of sEVs or exosomes provided herein comprises or is loaded with at least one polynucleotide comprising a gene encoding an AMPK protein, preferably AMPKα, preferably AMPKα1, most preferably AMPKα1-DN mutant protein, which gene is operably linked and under the control of SF1 promoter. Preferably, each of the sEVs of the population is loaded with at least one polynucleotide comprising a gene encoding an AMPK protein, preferably AMPKα, preferably AMPKα1, most preferably AMPKα1-DN mutant protein, which gene is operably linked and under the control of SF1 promoter. Preferably, the at least one polynucleotide is DNA, more preferably a plasmid. Thus, by designing a plasmid encoding AMPK-DN under the control of SF-1 (SF1-AMPK-DN plasmid), it is possible to express AMPK-DN only in SF1-expressing neurons located in the VMH. When the sEV or exosome is fused with the target cell, the AMPK protein, preferably AMPKα, preferably AMPKα1, most preferably AMPKα1-DN mutant protein, is expressed only if the target cell is an SF1-expressing neuron, preferably located in the ventromedial hypothalamus (VMH). Techniques used to load various cargos into sEVs include free-thaw cycles to fuse sEVs and liposomes, sonication, extrusion, permeabilization with saponin, and electroporation. Several commercial kits for loading nucleic acids into sEVs are available, and those skilled in the art will be familiar with them. Preferably, at least one polynucleotide, preferably a plasmid, is located mainly in the core of the sEV or exosome.

[0045] In one embodiment, the AMPK protein, preferably AMPKα, preferably AMPKα1, most preferably AMPKα1-DN mutant protein, contained in the polynucleotide of the sEV comprises at least the mutation or amino acid substitution D168A, and the amino acid numbering is expressed relative to the rat AMPKa1 protein, preferably relative to the wild type rat AMPKa1 sequence of SEQ ID NO: 40. In one embodiment, the nucleotide or amino acid sequence of the AMPK, AMPKα, AMPKα1, AMPKα1-DN protein is a rodent, preferably a murine sequence. In another embodiment, the nucleotide or amino acid sequence of the AMPK, AMPKα, AMPKα1, AMPKα1-DN sequence is a human homologue sequence, or a humanized or human codon optimized version of the murine sequence. In one embodiment, the AMPK alpha 1-DN mutant protein encoded by at least one polynucleotide contained in the sEV or exosome comprises, consists of, or consists essentially of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity over its entire length to SEQ ID NO: 1. In one embodiment, the at least one polynucleotide encoding the AMPK alpha 1-DN mutant protein comprises, consists of, or consists essentially of a polynucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity over its entire length to SEQ ID NO: 2.

[0046] In one embodiment, the steroidogenic factor 1 (SF1) polynucleotide sequence that is also contained in at least one polynucleotide contained in an sEV or exosome comprises, consists of, or consists essentially of a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:3 over its entire length.

[0047] In one embodiment, at least one polynucleotide encoding an AMPKα1-DN mutant protein operably linked and under the control of the SF1 promoter comprises, consists of, or essentially consists of a polynucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity to SEQ ID NO:4 over its entire length.

[0048] The sEV or exosome provided herein further comprises or is loaded, preferably primarily in its outer membrane, with a fusion protein comprising or consisting of a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b), the fusion protein comprising, consisting of or consisting essentially of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity over its entire length with SEQ ID NO:5. A "fusion protein" as used herein refers to a protein formed by at least two domains, which are linked one after the other such that they are synthesized or translated as a single unit, and thus the two domains of the fusion protein are part of a single polypeptide. In this particular case, the domains comprising or consisting of the fusion protein are the RVG peptide and the Lamp2b protein. In one embodiment, the domains of the fusion protein may be linked by a linker peptide. As used herein, a "linker peptide" is a short peptide sequence located between the domains of a fusion protein. The linker peptide is positioned to provide movement flexibility to the two domains contained in the fusion protein. In the context of the present invention, a linker peptide has at least one amino acid residue, preferably at least two consecutive amino acid residues, and optionally 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. Linker peptides include flexible linkers, rigid linkers, and in vivo cleavable linkers.

[0049] The population of sEVs or exosomes described herein is characterized in that it comprises at least one Lamp2b-RVG fusion protein. To that end, a Lamp2b-RVG plasmid, such as that designed as previously described in Reference 28 (see examples herein), can be transfected into exosome-producing cells, preferably exosome-producing immature dendritic cells, to transiently express a fusion protein comprising a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, preferably such a fusion protein comprises an amino acid sequence having at least 95% sequence identity over its entire length with SEQ ID NO:5. In one embodiment, the sEVs or exosomes further comprise a marker derived from the cells used to produce them (hereinafter referred to as producer cells). Preferably, the producer cells are immune cells, more preferably antigen-presenting cells, even more preferably dendritic cells. Preferably, the producer cells are mammalian cells. The producer cells can be primary or immortalized cells. In one embodiment, the producing cells are poorly immunogenic cells, preferably immature immune cells, preferably immature antigen presenting cells, most preferably immature dendritic cells or monocytes. "Immature" as used herein refers to cells that are not activated, biologically active, or do not present activation markers or molecules on their surface. Immature dendritic cells have a different morphological phenotype than mature dendritic cells. Immature dendritic cells have a round, smooth surface, whereas mature cells, such as mature dendritic cells, have a rough surface with multiple pseudopodia. Immature dendritic cells produce large amounts of exosomes that lack T cell activating factors, such as MHC-II, CD80, and CD86. Thus, in one embodiment, the producing cells, preferably immature dendritic cells, do not express activation markers, preferably T cell activating factor markers or molecules, such as major histocompatibility complex II (MHC-II), cluster of differentiation 80 (CD80), or cluster of differentiation 86 (CD86), or a combination thereof.In one embodiment, the producer cell is an immature immortalized cell, preferably an immature immortalized dendritic cell or monocyte, most preferably a JAWS II cell line derived from American Type Culture Collection CRL-1194; ATCC; Manassas, VA, USA, or a cell derived from said JAWS II cell line. Also preferably, the producer cell is a genetically modified cell that expresses an RVG-Lamp2b fusion protein, preferably in its outer membrane. In one embodiment, the producer cell is an immature antigen presenting cell characterized by having a statistically significant reduction in expression of at least one T cell activation factor molecule of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the expression of said T cell activation factor molecule in a mature antigen presenting cell. The reduction rate of said T cell activation factor molecule can be measured by techniques known in the art, for example by flow cytometry or RT-PCR.

[0050] The producing cells are grown in conditioned medium, and sEVs or exosomes are produced and released into said medium. In the context of the present invention, the term "conditioned medium" is understood as the supernatant of the cell culture. The population of sEVs or exosomes is released into the extracellular medium upon fusion of intracellular multivesicular bodies with the plasma membrane of the producing cells, so that the sEV or exosome outer membrane is similar to the one of the producing cells. Thus, in one embodiment, the population of sEVs or exosomes is positive for a cell marker, preferably a cell immature dendritic cell (iDC) marker. More preferably, the population of sEVs or exosomes comprises one or more specific exosome markers selected from the group consisting of ALIX, TSG101, CD9 or CD81, or any combination thereof. In another embodiment, the sEVs or exosomes are characterized by lacking MHC-II, CD80, CD86 or GRP94 markers, or any combination thereof. In one embodiment, since the sEVs or exosomes have an immature phenotype, they are less immunogenic.

[0051] In a preferred embodiment, the population according to the first aspect or any of its embodiments comprises a substantially pure population of immature dendritic cell (iDC)-derived sEVs or exosomes for use in treating or preventing obesity in a subject in need thereof, wherein each of the iDC-derived sEVs or exosomes, preferably the iDC-derived sEVs or exosomes, comprises at least one polynucleotide encoding an AMPK protein, preferably AMPKα, preferably AMPKα1, most preferably AMPKα1-DN mutant protein, operably linked to and under the control of the SF1 promoter, and wherein the iDC-derived sEVs or iDC-derived exosomes are engineered to transiently express at least one fusion protein comprising the RVG peptide fused to Lamp2b on their outer membrane.

[0052] As mentioned above, the results provide evidence that the population of sEVs or exosomes described herein can decrease or reduce the phosphorylation level of pACCα and / or the activation level of AMPK in SF1-expressing neurons located in the VMH. Furthermore, the present invention shows the remarkable specificity of the treatment disclosed herein, and when the phosphorylation level of pACCα was evaluated in other tissues, it was found that the pACCα level was not decreased in adjacent hypothalamic nuclei such as ARC, DMH and PVH (Figure 11), nor in other brain regions (cortex, thalamus, cerebellum, Figure 12A). This indicates that the activation level of AMPK in these brain regions was not altered by the treatment. Furthermore, to investigate the effects of the treatment in other tissues outside the brain and to elucidate how the systemic treatment may affect other biological processes such as testicular, adrenal and pituitary functions, circulating levels of testosterone and corticosterone (CORT) and mRNA expression of key steroidogenic enzymes in the testes and adrenal glands of mice treated with control and SF1-AMPKα1-DN-loaded sEVs were analyzed. The data showed that treatment with SF1-AMPKα1-DN loaded sEVs did not induce significant changes in any of these parameters (Figure 12d and Figure 12e). Similarly, no changes were observed in either circulating levels of luteinizing hormone (LH; Figure 12h) or mRNA levels of LHβ subunit (Figure 12i), whose pituitary production is known to be regulated by SF1. Overall, these results suggest that potential side effects of blunting AMPK signaling in other tissues (including those expressing SF1, such as pituitary, testes and adrenal glands) are likely negligible, confirming that activation levels of AMPK in said tissues were not affected or altered by the treatment, indicating that the treatment is not only effective but also safe and highly specific. Thus, the safety profile and high specificity of the treatment provided herein warrants its therapeutic use in the treatment of obesity.The safety profile and high specificity are relevant to the systemic administration of sEVs described herein, as this type of administration is less invasive than local (brain) administration, as shown in the Examples below, but the treatment is so specific that no off-target effects are seen, as the reduction in AMPK signaling is only present in the target tissue (SF1-expressing neurons located in the VMH region of the brain).

[0053] In view of this, in a preferred embodiment, the population of sEVs for use in the treatment of obesity according to the first aspect or any of its embodiments, when administered systemically, is capable of significantly reducing the activation level of AMPK, preferably AMPKa1, in SF1-expressing neurons located in the ventromedial hypothalamus (VMH) compared to the activation level of AMPK, preferably AMPKa1, in untreated SF1-expressing cells, preferably untreated SF1-expressing neurons, which reduction is not statistically significant in other SF1-expressing cells and / or tissues, preferably non-neural tissues / organs, more preferably those selected from the list consisting of the adrenal gland, testis, or pituitary gland. Thus, as a result of AMPK inhibition, the exogenous AMPKa1-DN isoform lacks kinase activity, and therefore the AMPK heterotrimer (α, β, γ; in this case, preferably α1) cannot be properly regulated and cannot exert its phosphorylation effect on downstream targets. "AMPK inhibition" is referred to herein as a reduction in the activation level of AMPK.

[0054] In one embodiment, the absence of a statistically significant decrease in other SF1-expressing cells and / or tissues, indicating specificity of treatment, may be due to the absence of those SF1-expressing cells and / or tissues in the subject, such as in the case of the absence of testes in females, and / or may be due to the treatment not affecting those cells / tissues despite their presence.

[0055] It should be noted that in addition to directly measuring the activation level of AMPK in SF1-expressing neurons located in the ventromedial hypothalamus (VMH), other surrogate markers showing a decrease in said activation level can also be used to test the safety and efficacy of the treatments provided herein. For example, the phosphorylation level of acetyl-CoA carboxylase alpha (pACCα) can be used as an indicator of the efficacy or safety of the treatment. Thus, in one embodiment, the population of sEVs for use in the treatment of obesity via a systemic route according to the first aspect or any of its embodiments, when administered systemically, can significantly decrease the phosphorylation level of pACCα in SF1-expressing neurons located in the ventromedial hypothalamus (VMH) compared to the phosphorylation level of pACCα in untreated SF1-expressing cells, preferably SF1-expressing neurons, and said decrease is not statistically significant in other SF1-expressing cells and / or tissues, preferably cells and / or tissues selected from the list consisting of the adrenal gland, testis, or pituitary gland.

[0056] "Statistically significant" or "significant" herein refers to the analyst's determination that the results of the data cannot be explained by chance alone. Statistical hypothesis testing is the way in which those skilled in the art make this determination. This test provides a p-value, which is the probability of observing a result as extreme as the results of the data, assuming that the result is truly due to chance alone. A p-value of 0.1 or less (preferably 0.05, 0.01, 0.001 or less) is considered statistically significant herein. For example, a decrease in pACCα levels or activation levels of AMPK protein in DF-expressing neurons located in the VMH is a statistically significant decrease or reduction when a statistical test is performed to compare treated SF1-expressing neurons located in the VMH with untreated SF1-expressing cells or neurons, and the resulting p-value of the statistical test is 0.1 or less, preferably 0.05, 0.01, 0.001 or less.

[0057] As used herein, "reduction" or "increase" refers to a decrease or increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, respectively, compared to a reference cell or value or reference tissue. Preferably, the increase is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20-fold increase relative to a reference cell or value or reference tissue. "Reference cell or tissue" as used herein refers to a cell or tissue that has not been treated with an exosomal sEV as described herein. Preferably, the cell or tissue is a mammalian cell, such as a human cell, preferably a neuron, or a cell or tissue from the central nervous system, preferably the VMH region. The reference cell can also be any SF1-expressing cell, preferably an SF1-expressing neuron located in the hypothalamus, preferably the VMH region. The reference cell can also be a single cell or a population of cells derived from the same subject or population of subjects. Reference cells can also be cells from subjects not suffering from obesity, preferably SF1 expressing cells. "Reference value" refers herein to the average known accurate measurement of a parameter, such as the level of pACCα or the activity level of AMPK protein in untreated or reference cells. Reference value is the average of repeated measurements from a more accurate measuring device. Those skilled in the art will know how to obtain said reference value.

[0058] Preferably, the SF1 expressing cell is a neuron located in the ventromedial hypothalamus (VMH). The phosphorylation or enzyme activity level can be expressed or measured by "absolute" quantification or "relative" or comparative quantification, which can be calculated by those skilled in the art using appropriate techniques. The methods or techniques used to measure the phosphorylation level of pACCα include, but are not limited to, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, flow cytometry, kinase activity assay, enzyme-linked immunosorbent assay (ELISA), and antibody array, mass spectrometry (MS). The methods or techniques used to measure the activity level of AMPK protein include, but are not limited to, optical, magnetic resonance and nuclear imaging methods (fluorescence-based methods, e.g., in situ zymography, magnetic resonance-based methods), mass spectrometry-based methods, sampling-based methods (microdialysis, electroosmosis-based methods), and enzyme activity measurement methods.

[0059] As explained above and shown in FIG. 4, mice treated with sEVs described herein showed a significant long-term reduction in body weight with no change in food intake. Notably, when treatment was discontinued, the effect of sEVs persisted, showing that treated mice showed no weight regain after treatment was discontinued (washout) when compared to control mice treated with control sEVs. Overall, these data indicate that the weight loss induced by a population of SF1-AMPKα1-DN loaded sEVs containing the RVG-Lamp2b fusion protein is not transient and treatment washout does not result in a rebound effect that causes treated mice to reach the weight of untreated mice. This data supports the idea that the treatments disclosed herein further provide an improvement or reduction in the rebound effect during and / or after washout or cessation of treatment. "Ameliorate" as used herein refers to an improvement in the patient's condition or an activity that seeks to correct or at least make a difficult to tolerate condition associated with the patient's condition more tolerable. In particular, the condition is obesity. "Rebound effect" is herein referred to as the production of negative symptoms when the effect of the treatment (population of sEVs or exosomes) has passed or the patient is no longer responding to it. As an example mode of rebound effect, refer to Figure 3 of the paper by Kawashima (2019, doi:10.3389 / fphys.2019.01483), where it is observed that after 40 days of the first cycle of high-fat diet, treated mice were significantly heavier than control mice. Thus, "rebound effect" is herein meant to mean an increase of at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% or more of the subject's basal body weight, where basal body weight is understood as the subject's body weight without treatment.

[0060] Preferably, the reduction or amelioration of the rebound effect is measured during or after the treatment washout for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or more than 90 days. Preferably, the reduction or amelioration of the rebound effect is measured during or after the treatment washout for at least 5-15, 5-20, 5-30, or 5-40 days.

[0061] Thus, in one embodiment, the population of sEVs or exosomes, when administered systemically, can significantly reduce or ameliorate the rebound effect, preferably measured as an increase of at least 15%-20% of the subject's basal body weight during and / or after no treatment, washout or cessation of treatment. In one embodiment, the population of sEVs or exosomes, when administered systemically, can significantly ameliorate or ameliorate obesity or its symptoms.

[0062] In some embodiments, the sEVs or exosomes are produced from autologous cells of the subject to be treated or obtained from cells isolated from the subject to be treated. In other embodiments, the sEVs or exosomes are produced from allogeneic cells to the subject to be treated or obtained from cells isolated from a donor other than the subject to be treated. In certain embodiments, the cells are mammalian cells, e.g., human cells. In certain embodiments, the cells are immature dendritic cells.

[0063] Preparation and storage of sEVs and their composition The populations described herein include an effective amount of an oil-in-water emulsion of a substantially pure population of sEVs or exosomes as defined above, together with one or more pharma- ceutically acceptable carriers. An "effective amount" is an amount sufficient to reduce, ameliorate, treat, and / or prevent obesity or rebound effects following obesity treatment. The effective amount will vary depending on several factors, including the age and weight of the subject being treated, how advanced the disease state is, the patient's general health, the severity of the symptoms, and whether the emulsion is administered alone or in combination with other therapies.

[0064] A carrier should be biologically acceptable without eliciting an adverse reaction (e.g., immune response) when administered to a host. Suitable pharma- ceutically acceptable carriers are well known in the art and vary depending on the desired form and mode of administration of the pharmaceutical formulation. For example, they may include diluents or excipients, such as fillers, binders, wetting agents, disintegrants, surfactants, lubricants, etc. Typically, the carrier is a solid, liquid, or vaporizable carrier, or a combination thereof. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the subject.

[0065] The population may be part of a composition that includes an emulsion containing sEVs or exosomes. Such emulsions may include oil-in-water emulsions. Emulsions may be made by means known in the art. They may be made from freshly prepared sEVs or exosomes, or lyophilized sEVs or exosomes, or sEVs or exosomes stored in oil. Emulsions are heterogeneous systems composed of at least two immiscible liquids, e.g., water and oil, one of which is uniformly dispersed as fine droplets throughout the other liquid phase, usually by a mechanical stirring process. The composition may further include at least one emulsifier. An emulsifier (also known as an "emulgent") is a substance that stabilizes an emulsion by increasing its kinetic stability.

[0066] sEVs or exosomes can be conveniently stored as a suspension or dispersion in oil. For this purpose, freshly prepared sEVs or exosomes or lyophilized exosomes can be suspended or dispersed in oil. The oil can include any suitable oil, such as vegetable oil. sEVs or exosomes can be suspended or dispersed in, for example, olive oil, palm oil, soybean oil or coconut oil. sEVs or exosomes can be suspended or dispersed in oil in any suitable ratio.

[0067] The suspension or dispersion of sEVs or exosomes in oil can be stored, for example, at room temperature, before use. The suspension or dispersion of sEVs or exosomes in oil can be such that the exosomes exhibit at least one biological activity of sEVs or exosomes following or after a storage period. The biological activity can include therapeutic activity, such as correcting, reducing, ameliorating, treating, preventing obesity or rebound effects after obesity treatment. The biological activity can be derived from the presence of dominant negative AMPKα1 protein (AMPKα1-DN), which reduces AMPK activity and pACC phosphorylation. The biological activity can be derived from a reduction in the phosphorylation level of acetyl-CoA carboxylase α (pACCα). The sEVs or exosomes may exhibit at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the biological activity defined above following or after storage.

[0068] In one embodiment, the composition is a pharmaceutical composition. The pharmaceutical composition is preferably enriched with a population of sEV-exosomes having a size of about 30-200 nm, preferably 30-150 nm. The amount of sEV or exosomes can be measured by protein amount, for example, by using Bradford assay (BioRad) or BCA protein assay kit (Pierce). However, the optimal dose is selected according to the route of administration, treatment regimen and / or administration schedule, taking into account existing toxicity and efficacy data. In a preferred embodiment, the substantially pure population of sEV or exosomes contained in the composition is at a dose that can provide a weight reducing or BAT increasing effect in the absence of toxic effects.

[0069] Furthermore, the population or composition or pharmaceutical composition is formulated to be compatible with its intended route of administration. As shown in the examples and discussed above, the route of administration can be systemic or local. Local routes of administration include intraventricular administration or, more preferably, specific stereotactic administration in individual hypothalamic nuclei. As used herein, "systemic route of administration" refers to the direct or indirect administration of the composition into the circulatory system. Systemic administration is usually safer than local administration, but may not be well tolerated or may cause off-target effects. However, the present invention demonstrates the safety of the treatment when administered systemically, and therefore, this is the preferred route of administration for the treatments provided herein. Systemic routes of administration include parenteral routes, such as intravascular, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, epidural, or other, as well as oral, nasal, ocular, or rectal. A preferred systemic route of administration is intravascular, understood herein as intravascular administration, typically including intravenous or intraarterial administration.

[0070] Methods for producing a population or composition or pharmaceutical composition according to the invention are known in the art and optionally include a step of specifically enriching a population of substantially pure sEVs or exosomes. For this purpose, generally any suitable method for purification and / or enrichment can be used, such as methods involving magnetic particles, filtration, dialysis, ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA) and / or chromatography.

[0071] As explained above, the main objective of the present invention is to provide a safe and effective tissue-specific population of sEVs for treating or preventing obesity. The use provided herein can be short-term or long-term, defined as a period of more than 3 months. The use provided herein for treating or preventing obesity minimizes side effects and / or complications, particularly side effects and / or complications resulting from suppressing the activity of AMPK protein in SF1-expressing tissues. Furthermore, the use provided herein can simultaneously treat other diseases or obesity-related diseases, such as type II diabetes, cardiovascular disease, hypertension or hypercholesterolemia.

[0072] The second aspect of the invention refers to a population of sEVs as defined under the first aspect, wherein the sEVs comprise at least one polynucleotide encoding an AMPK protein, preferably an AMPKα, preferably an AMPKα1, most preferably an AMPKα1-DN (dominant-negative AMP-activated protein kinase α1 mutant) protein, operably linked and under the control of the steroidogenic factor 1 (SF1) promoter, and the sEVs are engineered to transiently express in their outer membrane at least one fusion protein comprising a neurotrophic rabies virus (RVG) peptide fused to lysosomal-associated membrane protein 2b (Lamp2b). As the sEVs are characterized above under the first aspect of the invention, all embodiments relating to sEVs are herein also applicable to the second aspect.

[0073] Preferably, the population of sEVs is a pure population of exosomes, preferably exosomes. In one embodiment, the population of sEVs or exosomes is a substantially pure population. In some embodiments, the population is substantially pure or enriched in sEVs or exosomes, comprising at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of sEVs or exosomes. In one embodiment, the sEVs or exosomes are spherical or round. The size-related embodiments are defined above under the first aspect and apply herein.

[0074] The population of sEVs or exosomes provided herein comprises or is loaded with at least one polynucleotide comprising a gene encoding an AMPK protein, preferably AMPKα, preferably AMPKα1, most preferably an AMPKα1-DN mutant protein, which is operably linked and under the control of the SF1 promoter. Preferably, each of the sEVs of the population is loaded with at least one polynucleotide comprising a gene encoding an AMPK protein, preferably AMPKα, preferably AMPKα1, most preferably an AMPKα1-DN mutant protein, which is operably linked and under the control of the SF1 promoter.

[0075] In one embodiment, the AMPKα1-DN mutant protein encoded by at least one polynucleotide contained in an sEV or exosome comprises, consists of, or essentially consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1, 39 or 41 over its entire length.

[0076] In one embodiment, the AMPKα1-DN mutant protein encoded by at least one polynucleotide contained in the sEV or exosome comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, 99.2%, 99.4%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO: 40 (wild type rat AMPKα1 sequence), with the proviso that the amino acid residue at position 168 of SEQ ID NO: 40, which corresponds to aspartic acid, is replaced by an amino acid residue that is not aspartic acid, preferably by alanine (mutation D168A).

[0077] It should be noted that throughout this specification and all documents, the positions of amino acid residues are preferably numbered sequentially or consecutively from the N-terminus of the protein or polypeptide. It should also be noted that the amino acid numbering, and therefore the number of amino acid substitutions resulting in the AMPKα1-DN mutant protein defined herein, may vary within different AMPKα1 proteins. For example, the substitution D168A in SEQ ID NO: 40 or 41 is equivalent to the substitution D169A in SEQ ID NO: 1 (AMPKα1-DN mutant protein amino acid sequence with Myc-Tag and G-linker) and the substitution D156A in SEQ ID NO: 39 (AMPKα1-DN mutant protein amino acid sequence without Myc-Tag and G-linker). Therefore, it should be understood that, regardless of the numbering in which the aspartic acid is placed in the sequence of the AMPKα1 protein, the AMPKα1-DN mutant protein defined herein can be arrived at if it is changed for different amino acid residues. Preferably, the aspartic acid mutated in the sequence of AMPKα1 to generate the AMPKα1-DN mutant protein is the aspartic acid of the conserved sequence "NAKIADFGLS". Preferably, said AMPKα1-DN mutant protein is capable of impairing or reducing the activity of the endogenous functional counterpart (AMPKα1 wild type) of the cell, leading to a reduction in pACC phosphorylation levels in treated cells, preferably in the VMH region, but not in other SF-1 expressing tissues such as the adrenal gland, pituitary gland and testis.

[0078] As explained above in the first aspect, the sEVs or exosomes provided herein preferably further comprise or are loaded with a fusion protein, mainly in their outer membrane, comprising or consisting of a neurotrophic rabies virus (RVG) peptide fused to a lysosome-associated membrane protein 2b (Lamp2b), the fusion protein comprising, consisting of or consisting essentially of an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity with SEQ ID NO:5 over its entire length. In this case, the domains comprising or consisting of the fusion protein are the RVG peptide and the Lamp2b protein. In one embodiment, the domains of the fusion protein may be linked by a linker peptide.

[0079] In one embodiment, the population of sEVs according to the second aspect or any of its embodiments is for use in therapy, hi one embodiment, the population of sEVs according to the second aspect is for use in the treatment or prevention of obesity in a subject in need thereof, preferably wherein the obesity is diet-induced and / or genetically-induced obesity, preferably leptin receptor (LEPR) deficiency-induced obesity.

[0080] In one embodiment, the population of sEVs according to the second aspect or any of its embodiments is for use in ameliorating or reducing the rebound effect during and / or after washout of a treatment for obesity. The descriptions and embodiments of "ameliorating" and "rebound effect" are included above under the first aspect and apply herein.

[0081] In one embodiment, the population of sEVs, preferably when administered systemically, is capable of significantly reducing the activation level of AMP-activated protein kinase (AMPK) in SF1-expressing neurons located in the ventromedial hypothalamus (VMH) compared to the activation level of AMPK in untreated SF1-expressing cells, which is not significantly reduced in other SF1-expressing tissues selected from the list consisting of the adrenal gland, the testis, or the pituitary gland; the population of sEVs comprises at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKα1-DN) mutant protein, the amino acid sequence of the AMPKα1-DN mutant protein having at least 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1, 39 or 41; the AMPKα1-DN mutant protein is operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter, preferably the SF1 promoter has at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3; sEVs are engineered to transiently express in their outer membrane at least one fusion protein containing a neurotropic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, The population is for use in the treatment or prevention of obesity, preferably via a systemic route of administration, in a subject in need of such treatment or prevention.

[0082] In one embodiment, the population of sEVs, preferably when administered systemically, is capable of significantly reducing the activation level of AMP-activated protein kinase (AMPK) in SF1-expressing neurons located in the ventromedial hypothalamus (VMH) compared to the activation level of AMPK in untreated SF1-expressing cells, which is not significantly reduced in other SF1-expressing tissues selected from the list consisting of the adrenal gland, the testis, or the pituitary gland; The population of sEVs comprises at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKα1-DN) mutant protein, the amino acid sequence of which has at least 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% sequence identity with SEQ ID NO: 40 (wild-type rat AMPKα1 sequence), with the proviso that the amino acid residue at position 168 of SEQ ID NO: 40, which corresponds to aspartic acid, is replaced by an amino acid residue that is not aspartic acid, preferably by alanine (mutation D168A); the AMPKα1-DN mutant protein is operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter, preferably the SF1 promoter has at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3; sEVs are engineered to transiently express in their outer membrane at least one fusion protein containing a neurotropic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, The population is for use in the treatment or prevention of obesity, preferably via a systemic route of administration, in a subject in need of such treatment or prevention.

[0083] Sequence Listing SEQ ID NO:1: AMPKα1-DN mutant protein amino acid sequence. Note: (underline: Myc-Tag) (Grey highlight: G-linker) (Bold and underlined: mutation D168→A (amino acid numbering or position of mutation is that of rat AMPKα1 protein, preferably SEQ ID NO: 40)) TIFF2024530355000001.tif41170

[0084] SEQ ID NO:2: Polynucleotide sequence encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKalpha1) mutant protein. Note: Underlined (Myc-tag sequence) TIFF2024530355000002.tif129170

[0085] SEQ ID NO: 3: Steroidogenic factor 1 (SF1) polynucleotide sequence. TIFF2024530355000003.tif58170

[0086] SEQ ID NO: 4: Polynucleotide sequence encoding the AMPKα1-DN mutant protein operably linked and under the control of the SF1 promoter. TIFF2024530355000004.tif193170

[0087] SEQ ID NO: 5: Fusion protein: Neurotropic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b). Note: (No underline: Lamp2b sequence) (Underlined: RVG sequence) TIFF2024530355000005.tif36170

[0088] SEQ ID NO: 38: Nucleotide sequence of a fusion protein consisting of a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b). Note: (No underline: Lamp2b sequence) (Underlined: RVG sequence) TIFF2024530355000006.tif109170

[0089] SEQ ID NO: 39: AMPKα1-DN mutant protein amino acid sequence (without Myc-Tag peptide and G-linker) Bold and underlined: mutation D168->A (amino acid numbering or position of the mutation is with respect to the rat AMPKα1 protein, preferably that of SEQ ID NO: 40) Met Glu Lys Gln Lys His Asp Gly Arg Val Lys Ile Gly His Tyr Ile 1 5 10 15 Leu Gly Asp Thr Leu Gly Val Gly Thr Phe Gly Lys Val Lys Val Gly 20 25 30 Lys His Glu Leu Thr Gly His Lys Val Ala Val Lys Ile Leu Asn Arg 35 40 45 Gln Lys Ile Arg Ser Leu Asp Val Val Gly Lys Ile Arg Arg Glu Ile 50 55 60 Gln Asn Leu Lys Leu Phe Arg His Pro His Ile Ile Lys Leu Tyr Gln 65 70 75 80 Val Ile Ser Thr Pro Ser Asp Ile Phe Met Val Met Glu Tyr Val Ser 85 90 95 Gly Gly Glu Leu Phe Asp Tyr Ile Cys Lys Asn Gly Arg Leu Asp Glu 100 105 110 Lys Glu Ser Arg Arg Leu Phe Gln Gln Ile Leu Ser Gly Val Asp Tyr 115 120 125 Cys His Arg His Met Val Val His Arg Asp Leu Lys Pro Glu Asn Val 130 135 140 Leu Leu Asp Ala His Met Asn Ala Lys Ile Ala Ala Phe Gly Leu Ser 145 150 155 160 Asn Met Met Ser Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro 165 170 175 Asn Tyr Ala Ala Pro Glu Val Ile Ser Gly Arg Leu Tyr Ala Gly Pro 180 185 190 Glu Val Asp Ile Trp Ser Ser Gly Val Ile Leu Tyr Ala Leu Leu Cys 195 200 205 Gly Thr Leu Pro Phe Asp Asp Asp His Val Pro Thr Leu Phe Lys Lys 210 215 220 Ile Cys Asp Gly Ile Phe Tyr Thr Pro Gln Tyr Leu Asn Pro Ser Val 225 230 235 240 Ile Ser Leu Leu Lys His Met Leu Gln Val Asp Pro Met Lys Arg Ala 245 250 255 Thr Ile Lys Asp Ile Arg Glu His Glu Trp Phe Lys Gln Asp Leu Pro 260 265 270 Lys Tyr Leu Phe Pro Glu Asp Pro Ser Tyr Ser Ser Thr Met Ile Asp 275 280 285 Asp Glu Ala Leu Lys Glu Val Cys Glu Lys Phe Glu Cys Ser Glu Glu 290 295 300 Glu Val Leu Ser Cys Leu Tyr Asn Arg Asn His Gln Asp Pro Leu Ala 305 310 315 320 Val Ala Tyr His Leu Ile Ile Asp Asn Arg Arg Ile Met Asn Glu Ala 325 330 335 Lys Asp Phe Tyr Leu Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp 340 345 350 His His Leu Thr Arg Pro His Pro Glu Arg Val Pro Phe Leu Val Ala 355 360 365 Glu Thr Pro Arg Ala Arg His Thr Leu Asp Glu Leu Asn Pro Gln Lys 370 375 380 Ser Lys His Gln Gly Val Arg Lys Ala Lys Trp His Leu Gly Ile Arg 385 390 395 400 Ser Gln Ser Arg Pro Asn Asp Ile Met Ala Glu Val Cys Arg Ala Ile 405 410 415 Lys Gln Leu Asp Tyr Glu Trp Lys Val Val Asn Pro Tyr Tyr Leu Arg 420 425 430 Val Arg Arg Lys Asn Pro Val Thr Ser Thr Phe Ser Lys Met Ser Leu 435 440 445 Gln Leu Tyr Gln Val Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser 450 455 460 Ile Asp Asp Glu Ile Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gln 465 470 475 480 Arg Ser Gly Ser Ile Ser Asn Tyr Arg Ser Cys Gln Arg Ser Asp Ser 485 490 495 Asp Ala Glu Ala Gln Gly Lys Pro Ser Glu Val Ser Leu Thr Ser Ser 500 505 510 Val Thr Ser Leu Asp Ser Ser Pro Val Asp Val Ala Pro Arg Pro Gly 515 520 525 Ser His Thr Ile Glu Phe Phe Glu Met Cys Ala Asn Leu Ile Lys Ile 530 535 540 Leu Ala Gln 545

[0090] Figure 40: 5'-AMP conjugated to the α-1(RAT) norvegicus)]。NCBI Database:NP_062015.2 Put Arg Arg Leu Ser Ser Trp Arg Lys Put Ala Thr Ala Glu Lys Gln 1 5 10 15 Lys His Asp Gly Arg Val Lys Ile Gly His Tyr Ile Leu Gly Asp Thr 20 25 30 Leu Gly Val Gly Thr Phe Gly Lys Val Lys Val Gly Lys His Glu Leu 35 40 45 Thr Gly His Lys Val Ala Val Lys Ile Leu Asn Arg Gln Lys Ile Arg 50 55 60 Ser Leu Asp Val Val Gly Lys Ile Arg Arg Glu Ile Gln Asn Leu Lys 65 70 75 80 Leu Phe Arg His Pro His Ile Ile Lys Leu Tyr Gln Val Ile Ser Thr 85 90 95 Pro Ser Asp Ile Phe Met Val Met Glu Tyr Val Ser Gly Gly Glu Leu 100 105 110 Phe Asp Tyr Ile Cys Lys Asn Gly Arg Leu Asp Glu Lys Glu Ser Arg 115 120 125 Arg Leu Phe Gln Gln Ile Leu Ser Gly Val Asp Tyr Cys His Arg His 130 135 140 Met Val Val His Arg Asp Leu Lys Pro Glu Asn Val Leu Leu Asp Ala 145 150 155 160 His Met Asn Ala Lys Ile Ala Asp Phe Gly Leu Ser Asn Met Met Ser 165 170 175 Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro Asn Tyr Ala Ala 180 185 190 Pro Glu Val Ile Ser Gly Arg Leu Tyr Ala Gly Pro Glu Val Asp Ile 195 200 205 Trp Ser Ser Gly Val Ile Leu Tyr Ala Leu Leu Cys Gly Thr Leu Pro 210 215 220 Phe Asp Asp Asp His Val Pro Thr Leu Phe Lys Lys Ile Cys Asp Gly 225 230 235 240 Ile Phe Tyr Thr Pro Gln Tyr Leu Asn Pro Ser Val Ile Ser Leu Leu 245 250 255 Lys His Met Leu Gln Val Asp Pro Met Lys Arg Ala Thr Ile Lys Asp 260 265 270 Ile Arg Glu His Glu Trp Phe Lys Gln Asp Leu Pro Lys Tyr Leu Phe 275 280 285 Pro Glu Asp Pro Ser Tyr Ser Ser Thr Met Ile Asp Asp Glu Ala Leu 290 295 300 Lys Glu Val Cys Glu Lys Phe Glu Cys Ser Glu Glu Glu Val Leu Ser 305 310 315 320 Cys Leu Tyr Asn Arg Asn His Gln Asp Pro Leu Ala Val Ala Tyr His 325 330 335 Leu Ile Ile Asp Asn Arg Arg Ile Met Asn Glu Ala Lys Asp Phe Tyr 340 345 350 Leu Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp His His Leu Thr 355 360 365 Arg Pro His Pro Glu Arg Val Pro Phe Leu Val Ala Glu Thr Pro Arg 370 375 380 Ala Arg His Thr Leu Asp Glu Leu Asn Pro Gln Lys Ser Lys His Gln 385 390 395 400 Gly Val Arg Lys Ala Lys Trp His Leu Gly Ile Arg Ser Gln Ser Arg 405 410 415 Pro Asn Asp Ile Met Ala Glu Val Cys Arg Ala Ile Lys Gln Leu Asp 420 425 430 Tyr Glu Trp Lys Val Val Asn Pro Tyr Tyr Leu Arg Val Arg Arg Lys 435 440 445 Asn Pro Val Thr Ser Thr Phe Ser Lys Met Ser Leu Gln Leu Tyr Gln 450 455 460 Val Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser Ile Asp Asp Glu 465 470 475 480 Ile Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gln Arg Ser Gly Ser 485 490 495 Ile Ser Asn Tyr Arg Ser Cys Gln Arg Ser Asp Ser Asp Ala Glu Ala 500 505 510 Gln Gly Lys Pro Ser Glu Val Ser Leu Thr Ser Ser Val Thr Ser Leu 515 520 525 Asp Ser Ser Pro Val Asp Val Ala Pro Arg Pro Gly Ser His Thr Ile 530 535 540 Glu Phe Phe Glu Met Cys Ala Asn Leu Ile Lys Ile Leu Ala Gln 545 550 555

[0091] Figure 41: Figure 40: D168A Figure 5'-A MP was also expressed in the expression of α-1 [Rattus Norwegian)]。 Met Arg Arg Leu Ser Trp Arg Lys Met Ala Thr Ala Glu Lys Gln 1 5 10 15 Lys His Asp Gly Arg Val Lys Ile Gly His Tyr Ile Leu Gly Asp Thr 20 25 30 Leu Gly Val Gly Thr Phe Gly Lys Val Lys Val Gly Lys His Glu Leu 35 40 45 Thr Gly His Lys Val Ala Val Lys Ile Leu Asn Arg Gln Lys Ile Arg 50 55 60 Serving Asp Val Val Gly Lys With Arg Arg Glu And Gln Asn With Lys 65 70 75 80 Leu Phe Arg His Pro His Ile Lys Leu Tyr Gln Val Ile Ser Thr 85 90 95 Pro Ser Asp Ile Phe Met Val Met Glu Tyr Val Ser Gly Gly Glu Leu 100 105 110 Phe Asp Tyr Ile Cys Asn Gly Arg With Asp Glu Lys Glu Ser Arg 115 120 125 Arg Leu Phe Gln Gln Ile Leu Ser Gly Val Asp Tyr Cys His Arg His 130 135 140 Met Val Val His Arg Asp Leu Lys Pro Glu Asn Val Leu Leu Asp Ala 145 150 155 160 His Met Asn Ala Lys Ile Ala Ala Phe Gly Leu Ser Asn Met Met Ser 165 170 175 Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro Asn Tyr Ala Ala 180 185 190 Pro Glu Val Ile Ser Gly Arg Leu Tyr Ala Gly Pro Glu Val Asp Ile 195 200 205 Trp Ser Ser Gly Val Ile Leu Tyr Ala Leu Leu Cys Gly Thr Leu Pro 210 215 220 Phe Asp Asp Asp His Val Pro Thr Leu Phe Lys Lys Ile Cys Asp Gly 225 230 235 240 Ile Phe Tyr Thr Pro Gln Tyr Leu Asn Pro Ser Val Ile Ser Leu Leu 245 250 255 Lys His Met Leu Gln Val Asp Pro Met Lys Arg Ala Thr Ile Lys Asp 260 265 270 Ile Arg Glu His Glu Trp Phe Lys Gln Asp Leu Pro Lys Tyr Leu Phe 275 280 285 Pro Glu Asp Pro Ser Tyr Ser Ser Thr Met Ile Asp Asp Glu Ala Leu 290 295 300 Lys Glu Val Cys Glu Lys Phe Glu Cys Ser Glu Glu Glu Val Leu Ser 305 310 315 320 Cys Leu Tyr Asn Arg Asn His Gln Asp Pro Leu Ala Val Ala Tyr His 325 330 335 Leu Ile Ile Asp Asn Arg Arg Ile Met Asn Glu Ala Lys Asp Phe Tyr 340 345 350 Leu Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp His His Leu Thr 355 360 365 Arg Pro His Pro Glu Arg Val Pro Phe Leu Val Ala Glu Thr Pro Arg 370 375 380 Ala Arg His Thr Leu Asp Glu Leu Asn Pro Gln Lys Ser Lys His Gln 385 390 395 400 Gly Val Arg Lys Ala Lys Trp His Leu Gly Ile Arg Ser Gln Ser Arg 405 410 415 Pro Asn Asp Ile Met Ala Glu Val Cys Arg Ala Ile Lys Gln Leu Asp 420 425 430 Tyr Glu Trp Lys Val Val Asn Pro Tyr Tyr Leu Arg Val Arg Arg Lys 435 440 445 Asn Pro Val Thr Ser Thr Phe Ser Lys Met Ser Leu Gln Leu Tyr Gln 450 455 460 Val Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser Ile Asp Asp Glu 465 470 475 480 Ile Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gln Arg Ser Gly Ser 485 490 495 Ile Ser Asn Tyr Arg Ser Cys Gln Arg Ser Asp Ser Asp Ala Glu Ala 500 505 510 Gln Gly Lys Pro Ser Glu Val Ser Leu Thr Ser Ser Val Thr Ser Leu 515 520 525 Asp Ser Ser Pro Val Asp Val Ala Pro Arg Pro Gly Ser His Thr Ile 530 535 540 Glu Phe Phe Glu Met Cys Ala Asn Leu Ile Lys Ile Leu Ala Gln 545 550 555

[0092] The present invention also includes the following clauses.

[0093] 1. A population of small extracellular vesicles (sEVs) that, when administered systemically, is capable of significantly reducing the activation level of AMP-activated protein kinase (AMPK) in SF1-expressing neurons located in the ventromedial hypothalamus (VMH) compared to the activation level of AMPK in untreated SF1-expressing cells, but not in other SF1-expressing tissues selected from the list consisting of the adrenal gland, the testis, or the pituitary gland; the population of small extracellular vesicles (sEVs) is characterized in that it comprises at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKα1-DN) mutant protein operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter, the sEVs being engineered to transiently express in their outer membrane at least one fusion protein comprising a neurotrophic rabies virus (RVG) peptide fused to lysosomal-associated membrane protein 2b; The composition is for use in treating obesity via a systemic route of administration in a subject in need thereof. A population of small extracellular vesicles (sEVs).

[0094] 2. The population for use according to clause 1, wherein the treatment further provides an improvement or reduction in rebound effect during and / or after washout of the treatment.

[0095] 3. The population for use of any one of clauses 1 or 2, wherein the dominant negative AMP-activated protein kinase alpha 1 (AMPKalpha1) mutant protein encoded by at least one polynucleotide comprises an amino acid sequence having at least 95% sequence identity over its entire length to SEQ ID NO:1.

[0096] 4. The population for use according to any one of clauses 1 to 3, wherein at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKα1) mutant protein comprises a polynucleotide sequence having at least 95% sequence identity over its entire length to SEQ ID NO:2.

[0097] 5. The collection for use according to any one of clauses 1 to 4, wherein the steroidogenic factor 1 (SF1) promoter comprises a polynucleotide sequence having at least 95% sequence identity over its entire length to SEQ ID NO:3.

[0098] 6. The population for use according to any one of clauses 1 to 5, wherein at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase alpha 1 (AMPKα1) mutant protein operably linked and under the control of the steroidogenic factor 1 (SF1) promoter comprises a polynucleotide sequence having 100% sequence identity over its entire length to SEQ ID NO:4.

[0099] 7. The population for use according to any one of clauses 1 to 6, wherein the transiently expressed fusion protein comprising a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises an amino acid sequence having at least 95% sequence identity over its entire length to SEQ ID NO:5.

[0100] 8. The population for use according to any one of clauses 1 to 7, wherein the small extracellular vesicles have a size distribution of 30 to 150 nm.

[0101] 9. The population for use according to any one of clauses 1 to 8, wherein the small extracellular vesicles further comprise one or more specific markers selected from the group consisting of ALIX, TSG101, CD9 or CD81, or any combination thereof.

[0102] 10. The population for use according to any one of clauses 1 to 9, wherein the small extracellular vesicles are characterized by lacking the GRP94 marker.

[0103] 11. The population for use according to any one of clauses 1 to 10, wherein the small extracellular vesicles are produced or obtained from immature antigen-presenting cells, characterized in that the expression of at least one T cell activation factor molecule is statistically significantly reduced compared to the expression of said T cell activation factor molecule in mature antigen-presenting cells.

[0104] 12. The population for use according to any one of clauses 1 to 11, wherein the antigen presenting cells are dendritic cells, preferably a JAWS II cell line, and the at least one T cell activator molecule is one or more T cell activator molecules selected from the group consisting of major histocompatibility complex II (MHC-II), cluster of differentiation 80 (CD80) or cluster of differentiation 86 (CD86), or a combination thereof.

[0105] 13. The population for use according to any one of clauses 1 to 12, wherein the small extracellular vesicles are exosomes.

[0106] 14. The population for use according to any one of clauses 1 to 13, wherein the systemic route is an intravascular route.

[0107] 15. The population for use according to any one of clauses 1 to 14, wherein the treatment comprises reversing or ameliorating obesity. EXAMPLES

[0108] Example 1 method cell culture The JAWS II dendritic cell line was purchased from the American Type Culture Collection (CRL-1194; ATCC; Manassas, VA, USA). JAWS II cells were grown in a 37°C, 5% CO2 incubator in complete culture medium consisting of alpha-minimal essential medium (αMEM) (Lonza; Basel, Switzerland) containing ribonucleosides, desoxyribonucleosides and supplemented with 20% (vol / vol) fetal bovine serum (FBS) (Gibco, Life Technologies; Grand Island, NY, USA), 4 mM L-glutamine (Lonza; Basel, Switzerland), 1 mM sodium pyruvate (Lonza; Basel, Switzerland), 1% penicillin / streptomycin (GE Healthcare; Little Chalfont, Buckinghamshire, UK), and 5 ng / mL mouse GM-CSF (Miltenyi Biotec; San Diego, CA, USA). The mouse hypothalamic GT1-7 cell line (kindly provided by Eduardo Dominguez; University of Santiago de Compostela) was cultured in a 37°C incubator with 5% CO in complete medium consisting of Dulbecco's modified Eagle's medium (DMEM) (Lonza; Basel, Switzerland) containing 4.5 g / L glucose, 1 mM sodium pyruvate and 1 mM L-glutamine, and supplemented with 10% FBS (Gibco, Life Technologies; Grand Island, NY, USA) and 1% penicillin / streptomycin (GE Healthcare; Little Chalfont, Buckinghamshire, UK).The mouse neuroblastoma Neuro-2A cell line (kindly provided by the laboratory Micro et Nanomédecines Translationnelles, University of Angers, France) was grown at 37 °C and 5% CO2 in growth medium consisting of DMEM supplemented with 10% (vol / vol) FBS, 10 U / mL penicillin, 100 µg / mL streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate. Immortalized brown adipocytes derived from C57BL / 6J mice49 were seeded at a density of 2.5 × 105 cells / well in 6-well plates in DMEM containing 10% FBS, 20 mM HEPES, 1 nM T3 and 20 nM insulin until reaching 70%–80% confluence. Then, 500 nM dexamethasone, 1 μM rosiglitazone, 125 μM indomethacin, and 500 μM 3-isobutyl-1-methylxanthine (IBMX) were added for 44 h. The cells were then cultured in medium containing only T3 and insulin until differentiation (4-5 days). Primary cortical astrocytes were obtained from the cerebral cortex of 3-day-old C57BL / 6 mice and maintained in culture for 4-6 days. The cells were cultured in DMEM supplemented with 10% FBS.

[0109] animal Adult (8–12 weeks) male C57BL / 6 mice (25 g; Centro de Biomedicina Experimental; Santiago de Compostela, Spain or Jackson Laboratory, USA), nude mice (NRj:NMRI-Foxn1nu / Foxn1nu; Janvier Labs; Saint Berthevin, France) and C57BL / 6 homozygous UCP1 knockout (UCP1-KO; ucp1− / −) males and their corresponding wild-type littermates (bred at GTH University of Lubeck, Germany) were used for the experiments. Experiments were performed in accordance with the International Law on Animal Experimentation and approved by the USC Ethics Committee (project ID 15010 / 14 / 006) and the University of Iowa Animal Research Committee (protocol 8101549). Animals were housed under an artificial 12-h light (8:00 to 20:00) / 12-h dark cycle under controlled temperature and humidity conditions and were provided with a regular chow diet or a 60% HFD (D12492; Research Diets, Inc; New Brunswick, USA) and filtered tap water ad libitum for 10 weeks. For all procedures, animals were caged individually and acclimated to handling procedures under stress-free conditions. Experiments were performed in accordance with the International Law on Animal Experimentation and approved by the USC Ethics Committee (15012 / 2020 / 010).

[0110] Plasmids The Lamp2b-RVG plasmid was designed as previously described. Briefly, a plasmid encoding the Lamp2b sequence (kindly provided by Seow Yiqi, University of Oxford) containing NheI and BamHI restriction sites was cloned into the pEGFP-C1 backbone, taking care to remove the eGFP coding sequence. The RVG primer (forward: 5'-TCG ATA CAC CAT TTG GAT GCC CGA GAA TCC GAG ACC AGG GAC ACC TTG TGA CAT TTT TAC CAA TAG CAG AGG GAA GAG AGC ATC CAA CGG GT-3'; reverse: 5'-CCG GAC CCG TTG GAT GCT CTC TTC CCT CTG CTA TTG GTA AAA ATG TCA CAA GGT GTC CCT GGT CTC GGA TTC TCG GGC ATC CAA ATG GTG TA-3') was inserted between the XhoI and BspEI sites at the N-terminus of Lamp2b. A plasmid encoding a dominant-negative mutant of AMPKα1 (SF1-AMPKα1-DN) under the control of the steroidogenic factor-1 (SF1) promoter was purchased from Viraquest (North Liberty, IA, USA).

[0111] Generation and isolation of small extracellular vesicles JAWS II cells were cultured at 5 × 10 in a T75 cell culture flask in complete culture medium (see composition above) the day before transfection. 6Cells were seeded at a density of 1000 x 1000 cells / well. On the day of transfection, JAWS II cells were transiently transfected with the Lamp2b-RVG plasmid by MacsFectin transfection reagent (Miltenyi Biotec; San Diego, CA, USA). As indicated in the manufacturer's protocol, 20 μg of Lamp2b-RVG plasmid diluted in 350 μL of serum-free medium was added to 40 μL of MacsFectin diluted in 350 μL of serum-free medium. The mixture was incubated at room temperature for 20 min to allow the formation of transfection complexes before adding to the cells. After 24 h, the cell medium was replaced with FBS-sEV-free medium (complete αMEM supplemented with FBS ultracentrifuged twice at 200,000 g). After 48 h, the cell medium was collected and centrifuged twice for 10 min at 300 g and 2,000 g to remove cells and cell debris, respectively. The resulting supernatant was centrifuged at 20,000g for 30 min to exclude large EVs. The sEV pellet was further isolated from the large EV-depleted supernatant by a 200,000g centrifugation step for 2 h at 4°C using an MLA-50 rotor in an Optima Max-XP ultracentrifuge (Beckman Coulter; Brea, CA, USA). The sEV pellet was washed once with PBS using the same prior ultracentrifugation process and then resuspended in PBS. The sEV samples were kept at -80°C until use.

[0112] Electron microscopy sEVs were fixed overnight in freshly prepared 2.5% paraformaldehyde (PFA) in 0.1 M sodium cacodylate buffer (pH 7.4). sEVs were pelleted by the ultracentrifugation process described above and resuspended in 2.5% glutaraldehyde solution. sEVs were deposited on copper grids, negatively stained with phosphotungstic acid, and observed at 200 kV with a JEM1400 transmission electron microscope (JEOL; Peabody, MA, USA).

[0113] Nanoparticle Tracking Analysis (NTA) 50 μg of purified, (i) native unmodified, (ii) Lamp2b-RVG neuron-targeted or (iii) Lamp2b-RVG SF1-AMPKα1-DN-loaded sEVs were diluted in 1 mL of PBS and analyzed for size distribution at 37°C using a NanoSight NS 300 (Malvern Instruments; Orsay, France) according to the manufacturer's instructions.

[0114] Briefly, NTA applies both the light scattering and Brownian motion characteristics of the particles of interest to obtain the size distribution of nanoparticles in a liquid suspension. A 60 s video was recorded and then analyzed by the NTA software, which determines the size distribution using the Stokes-Einstein equation.

[0115] Assessment of small extracellular vesicle loading and nucleic acid content Purified sEVs were loaded with nucleic acids - either siRNA Texas Red (System Biosciences; Palo Alto, CA, USA), GFP plasmid or SF1-AMPKα1-DN - using Exo-Fect (System Biosciences) according to the manufacturer's protocol. Briefly, sEVs (50-300 μg) were incubated with 10 μL of Exo-Fect solution, 20 pmol of siRNA Texas Red or 5 μg of plasmid (GFP or SF1-AMPKα1-DN) and 70 μL of PBS at 37 °C for 10 min.

[0116] Then, 30 μL of Exo-Quick solution (System Biosciences; Palo Alto, CA, USA) was added, and the mixture was placed at 4° C. for 30 min. Samples were then centrifuged at 14,000 rpm for 3 min to pellet the sEVs, which were then resuspended in an appropriate volume of PBS depending on their subsequent use. The sEVs were either used directly or stored at −80° C. until further use.

[0117] To evaluate loading with SF1-AMPKα1-DN plasmid, purified SF1-AMPKα1-DN loaded sEVs were treated or not with DNase I (RNase free, Qiagen, Valencia, CA, USA) diluted in RDD buffer (according to manufacturer's instructions) for 10 min at 37°C in the absence or presence of 0.2% TritonX-100 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). To inactivate DNase activity, samples were heated at 75°C for 10 min. Both undigested and DNA digested sEVs were then subjected to end-point PCR in the presence of AMPK or GAPDH (as control) primers (sequences below).

[0118] The products of the PCR reactions were then run on a 2% agarose gel (Sigma-Aldrich; St. Louis, MO, USA) containing 0.001% ethidium bromide. Gels were visualized with UV light in an INFINITY VX2 1120M gel documentation system (Vilber Lourmat; Collegien France). AMPK: forward: 5'-ACG GCC GAG AAG CAG AAG CAC-3'; reverse: 5'-TCG TGC TTG CCC ACC TTC AC-3'; GAPDH: forward: 5'-AGT ATG TCG TGG AGT CTA C-3'; reverse: 5'-CAT ACT TGG CAG GTT TCT C-3'.

[0119] Labeling and biodistribution analysis of small extracellular vesicles sEVs were stained with DID solution (excitation maximum: 644 nm; emission maximum: 665 nm) (Vybrant™ DiD Cell, Molecular Probes; Eugene, OR, USA) according to the manufacturer's protocol. Briefly, sEVs were incubated with 5 μg / mL Vybrant™ DiD Cell in PBS for 10 min at room temperature and washed in PBS with two 200,000 g ultracentrifugation steps for 2 h. The resulting DID-labeled sEVs were collected in PBS and used directly.

[0120] Nude mice were intravenously injected with 100 μg of DID-labeled native or Lamp2b-RVG sEVs. A multispectral imaging system, MAESTRO In-Vivo Fluorescence Imaging System (Cambridge Research&Instrumentation; Woburn, MA, USA), was used to analyze the biodistribution of DID-labeled sEVs. The biodistribution of DID-labeled sEVs was analyzed at different times (30 min, 2, 4, and 6 h) in mice sedated with isoflurane. After the animals were sacrificed, the fluorescence of each of the isolated organs (liver, spleen, lung, heart, brain, and skeletal muscle) was also analyzed. The data were analyzed by MAESTRO In-Vivo Fluorescence Imaging System software (Cambridge Research&Instrumentation).

[0121] In vitro treatment with sEVs Twenty-four hours prior to treatment with sEVs, (i) JAWS II cells were plated at 2 × 10 in a μ-Slide 8Well (Ibidi; Munich, Germany) containing 300 μL of complete growth medium. 4 (ii) GT1-7 and (iii) Neuro2A cells were seeded at a density of 2 × 10 in 1 mL of complete growth medium. 5 (iv) primary cortical astrocytes were seeded into 6-well plates at a density of 5 × 10 cells in 1 mL of complete growth medium. 5On the day of treatment, JAWS II cells were incubated with non-loaded sEVs, siRNA Texas Red or GFP-loaded sEVs (1 μg for all conditions) before fixation and the respective fluorescence - siRNA Texas Red (excitation maximum: 596 nm and emission maximum: 615 nm) and GFP (excitation maximum: 488 nm and emission maximum: 509 nm) - was assessed at different times (2, 6 and 24 h) by confocal microscopy (CLMS700, Zeiss, ZEN fluorescence; Jena, Germany). On the other hand, GT1-7, Neuro2A cells, brown adipocytes and primary cortical astrocytes were treated with 10 μg / mL SF1-AMPKα1-DN-loaded sEVs for 24 h and then harvested and proteins were extracted for further analysis.

[0122] Stereotaxic treatment with sEVs DIO mice were placed in a stereotaxic frame (David Kopf Instruments; Tujunga, CA, USA) under ketamine-xylazine anesthesia (50 mg / kg, intraperitoneally).

[0123] The VMH was targeted bilaterally with a 32-gauge needle (Hamilton; Reno, NV, USA) using the following stereotaxic coordinates; 17、18 As shown in Fig. 1, the tumor was 1.7 mm posterior to the bregma, ±0.5 mm lateral to the midline, and 5.5 mm deep. 2 μg of control or SF1-AMPKα1-DN-loaded sEVs were injected at 100 nL min -1 (0.5 μL at each injection site) for 10 min. Daily measurements of body weight, food intake, and BAT temperature (see below) were taken. On day 3, animals were sacrificed and organs were collected for further analysis (see below).

[0124] Systemic treatment with sEVs 100 μg of SF1-AMPKα1-DN unloaded or loaded sEVs (indirect quantification by considering the total sEV protein content measured by Bradford) were injected into the tail vein of mice for 3 days, respectively, for the corresponding time depending on the experiment. For time course experiments (both SF1-AMPKα1-DN plasmid in vivo expression and UCP1 BAT expression over time), mice were injected once and sacrificed at the corresponding time points (24 h, 48 h, 72 h and 1 week). Daily measurements of body weight, food intake and BAT temperature (see below) were performed. At the end of the different experimental procedures, animals were sacrificed and organs were collected for further analysis (see below). 6-15 animals per group were used for each experiment, which was repeated 2-6 times. The β3-AR specific antagonist SR59230A ([3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol-oxalate] (3 mg / Kg / day dissolved in DMSO; Tocris Bioscience; Bristol, UK) was administered subcutaneously twice daily at the start of the cycle at 8:00 and 20:00, starting 3 days before the first intravenous injection. For thermoneutral experiments, mice were housed in thermoneutral conditions (30 °C). Mice were adapted to temperature fluctuations (2 °C increase daily for 4 days) before the experimental procedures and daily measurements for 6 days as previously described.

[0125] temperature measurement Skin temperatures surrounding the BAT and the temperature at the base of the tail were recorded with an infrared camera (B335: Compact-Infrared-Thermal-Imaging-Camera; FLIR; West Malling, Kent, UK) and analyzed with a specific software package (FLIR-Tools-Software; FLIR; West Malling, Kent, UK) as described in 12,13,15-18,44. In all cases, the average temperature of a selected area was chosen. The size and landmarks of the area were similar for all mice. The emissivity (ε) was set to 0.95. To obtain consistency in the results analysis, BAT and tail temperature recordings were performed daily, always at the same time during all experimental procedures. Results are presented as time courses to highlight time-dependent effects or as the average of the different daily measurements performed throughout the experiment.

[0126] indirect calorimetry Animals were cultured according to our previous report. 13、15~18、44、51 EE, oxygen consumption (VO2), respiratory quotient (RQ) and locomotor activity (LA) were analyzed using a calorimetric system (LabMaster; TSE Systems; Bad Homburg, Germany) as described previously.

[0127] Nuclear magnetic resonance All studies were performed in a 9.4 T horizontal bore magnet (Bruker BioSpin, Ettlingen, Germany) with a gradient of 440 mT / m. For body composition, a quadrature volume coil (7 cm diameter) was used. NMR procedures were performed under sevoflurane anesthesia (6% induction and 3.5% maintenance in a gas mixture of 70% NO2 and 30% O2). During MRI studies, each animal was fixed in a Plexiglas holder using tooth bars, ear bars, and adhesive tape to minimize spontaneous movement during image acquisition. For body composition studies, fast low angle shot (FLASH) sequences with repetition time / echo time (RT / ET) = 1300 / 3.5 ms, number of averages (NA) = 2, 30 coronal slices of 1 mm, field of view (FOV) = 60 × 80 mm, and matrix size = 256 × 350 (in-plane resolution of 0.234 × 0.229 mm / pixel) were acquired with and without the fat suppression option to generate both “fat suppressed” and “fat” image sets.

[0128] Total acquisition time was 31 min. MR post-processing was performed using ImageJ software (W. Rasband, NIH, USA). Semi-automated image processing was used to create fat masks (total (total AT), subcutaneous adipose tissue (scAT) and visceral adipose tissue (vAT) volumes) comparing identical registered image sets with and without fat suppression option. MRI volumes were converted to weights using standard densities of adipose tissue (0.9 g / mL) and other tissues (1.04 g / mL). 52-54 .

[0129] Positron Emission Tomography-Computed Tomography Whole-body microPET / CT (positron emission tomography-computed tomography) images were acquired using an Albira PET / CT Preclinical Imaging System (Bruker Biospin; Woodbridge, CT, US). Mice received 2 -18 F-fluoro-2-deoxy-2-glucose ( 18 F-FDG) injection into the tail vein. 18Acquisition was performed 45 ± 10 min after F-FDG injection. Images were generated using the Bruker Albira Suite software version 5.0. Brown fat and liver regions were delineated by generating a 3-dimensional spherical volume of interest with a radius of 6 mm using image tools implemented in the AMIDE software (http: / / amide.sourceforge.net / ). Thus, the mean standardized uptake value (SUV) was calculated. 17、18 .

[0130] Sympathetic nerve activity (SNA) recording Multifiber recording of the SNA was performed as previously described. 12、15、17、18、55, obtained from the nerve subserving BAT. Each mouse was anesthetized with an intraperitoneal administration of ketamine (91 mg / kg body weight) and xylazine (9.1 mg / kg body weight) and intubated with PE-50 to provide an unobstructed airway for the mouse to spontaneously breathe O2-enriched room air. Next, a microrenathan tubing (MRE-40, Braintree Scientific; Braintree, MA, USA) was inserted into the right jugular vein for infusion of the continuous anesthetic α-chloralose (initial dose: 12 mg / kg, then a continuous dose of 6 mg / kg / h). Another MRE-40 catheter was inserted into the left carotid artery for continuous measurement of arterial pressure and heart rate. Core body temperature was monitored using a rectal probe and maintained continuously at 37.5 °C. Each mouse was then instrumented with a direct multi-fiber SNA from the nerve serving the subscapular BAT. A bipolar platinum-iridium electrode (36 gauge, AM Systems; Sequim, WA, USA) was suspended under the nerve and secured with silicone gel (Kwik-Sil, WPI; Sarasota, FL, USA). The electrode was attached to a high impedance probe (HIP-511, Grass Instruments; West Warwick, RI, USA) and the nerve signal was amplified 105 times and filtered with a Grass P5 AC preamplifier at 100 Hz and 1,000 Hz cutoffs. The amplified and filtered nerve signal was routed to a speaker system and an oscilloscope (model 54501A, Hewlett-Packard; Palo Alto, CA, USA) to monitor the audio and visual quality of the BAT sympathetic nerve recordings for quantification purposes. The amplified and filtered neural signal was also directed to a MacLab analog-to-digital converter (model 8S, ADInstruments; Colorado Springs CO, USA) and reset voltage integrator (model B600C, University of Iowa Bioengineering; Iowa City, IA, USA) containing software (MacLab Chart Pro, version 7.0; Takoma, MD, USA) that utilizes cursors to analyze the number of spikes per second above a background noise threshold.

[0131] Under stable isothermal (37.5°C) conditions and under anesthesia, baseline BAT SNA was recorded for 30 min. The nerve was then cut distally and efferent SNA was recorded for an additional 15 min. Background noise was then measured by recording remaining activity after cutting the nerve proximal to the recording site, and this was subtracted to measure actual SNA. Afferent nerve activity was determined by subtracting efferent from total nerve activity. Systolic, diastolic, and mean arterial pressures along with heart rate were measured in all animals during nerve recordings.

[0132] Sample processing Mice were killed by dislocation and decapitation. From each animal, VMH, cortex, thalamus and cerebellum as well as peripheral tissues were collected for Western blotting and real-time RT-PCR and immediately homogenized on ice to preserve RNA and protein levels. These samples and serum were stored at -80°C until further processing. Dissection of the VMH was performed as previously described. 12、13、15~18、44 , performed by a micropunch procedure under a microscope.

[0133] blood biochemistry LH serum levels were measured in duplicate using double antibody technique and radioimmunoassay kits provided by Dr. AF Parlow (National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Torrance, CA) as described in detail elsewhere. Serum testosterone and CORT levels were measured using RIA kits (MP Biomedicals, LLC; Santa Ana, CA, USA). Leptin circulating levels were measured using a mouse ELISA kit (#EZML-82K, Millipore; Billerica, MA, USA). Cholesterol (#1001093, Spinreact; Barcelona, ​​Spain), triglycerides (#1001314, Spinreact; Barcelona, ​​Spain), free fatty acids (NEFA Standard: 270-77000 and NEFA-HR R2 set: 436-91995 WAKO; Neuss, Germany) circulating levels as well as AST (#41272, AST) and ALT (#41282, ALT) activities (Spinreact; Barcelona, ​​Spain) were measured by spectrophotometry on a Multiskan GO spectrophotometer (Invitrogen-Thermofisher Carlsbad, CA, USA). GDF15 serum levels were measured using a mouse ELISA kit (Cloud Clone Corp., Wuhan, China).

[0134] Serum cytokines (IL-6 and IP-10) were measured using Milliplex kits (Merk-Millipore, France) according to the manufacturer's instructions.

[0135] RT-PCR analysis For testis and adrenal gland analysis, real-time PCR (SYBR GreenER™ qPCR SuperMix System; Invitrogen; Carlsbad, CA, USA) was performed as previously described57,58 using the following specific primers:· STAR: forward: 5'-AGT TCG ACG TCG GAG CTC TCT-3'; reverse: 5'-TAC TTA GCA CTT CGT CCC CG-3';· P450scc: forward: 5'-GAT TGC GGA GCT GGA GAT GA-3'; reverse: 5'-TCT TTT CTG GTC ACG GCT GG-3';· 17β-HSD3: forward: 5'-CTG AGC ACT TCC GGT GAG AG-3'; reverse: 5'-GGC CTT TCC TCC TTG ACT CC-3';· LHβ: forward: 5'-GAG TTC TGC CCA GTC TGC AT-3';Reverse:5'-AGG AAA GGA GAC TAT GGG GTC T-3'·S11:Forward:5'-CAT TCA GAC GGA GCG TGC TTA C-3'; Reverse: 5'-TGC ATC TTC ATC TTC GTC AC-3'.

[0136] For RNA levels of skeletal muscle thermogenic markers, real-time PCR (TaqMan; Applied Biosystems; Foster City, CA, USA) was performed using the following specific primers and probes: Atp2a2: forward: 5'-TCC GCT ACC TCA TCT CAT CC-3'; reverse: 5'-CAG GTC TGG AGG ATT GAA CC-3'; Gdp2: forward: 5'-GAA GGG GAC TAT TCT TGT GGG T-3'; reverse: 5'-GGA TGT CAA ATT CGG GTG TGT-3'; Pparγ: forward: 5'-TCG CTG ATG CAC TGC CTA TG-3'; reverse: 5'-GAG AGG TCC ACA GAG CTG ATT-3'; Ryr1: forward: 5'-CAG TTT TTG CGG ACG GAT GAT-3'; reverse: 5'-CAC CGG CCT CCA CAG TAT TG-3'; Sln: forward: 5'-GAG GTG GAG AGA CTG AGG TCC TTG G-3'; reverse: 5'-GAA GCT CGG GGC ACA CAG CAG-3'; Ucp3: forward: 5'-GAG ATG GTG ACC TAC GAC ATC A-3'; reverse: 5'-GCG TTC ATG TAT CGG GTC TTT A-3'.

[0137] For analysis of SF1-AMPKα1-DN expression in vivo, total RNA was isolated from tissues from mice sacrificed at the corresponding times with Trizol (Invitrogen; Carlsbad, CA, USA) and then reverse transcribed into cDNA using M-MLV enzyme (Invitrogen; Carlsbad, CA, USA) according to the supplier's protocol.

[0138] The corresponding cDNA was used as template in PCR reactions containing 10 μM of each primer (see sequences below). PCR cycle conditions were designed as follows: an initial denaturation step at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. PCR products were analyzed on a 2% agarose gel. The following primers were used: SF1-AMPKα1-DN: forward: 5'-AAA CAC CAA GGC GTA CGG AA-3'; reverse: 5'-TGG CGG CCG CTC TAG ATT AC-3'; HPRT: forward: 5'-GGT TAA GCA GTA CAG CCC CA-3'; reverse: 5'-TCC AAC ACT TCG AGA GGT CC-3'.

[0139] AMPK activity assay AMPK activity was measured using the CycLex AMPK Kinase Assay (CY-1182; MBL International Corporation; Woburn, MA, US) according to the manufacturer's recommendations and those suggested by others. Briefly, 10 ul of lysis buffer containing 5 μg of VMH protein was added to 90 μL of kinase assay buffer. Each sample was analyzed in duplicate, and U2OS WT or AMPK KO cell extracts were used as controls. Absorbance was measured at 450 / 550 nm on a MultiSkan Go (Invitrogen-Thermofisher; Carlsbad, CA, USA).

[0140] immunohistochemistry Brains were post-fixed overnight in 4% PFA at 4°C, equilibrated in a solution containing 30% sucrose in Tris-buffered saline (TBS, pH 7.2), and sectioned into 30 μm coronal slices on a cryostat (CM3050S, Leica; Wetzlar, Germany). Brain slices along the medial part of the mediobasal hypothalamus were selected. Brain sections were first washed with TBS, blocked with SUMI solution (0.25% porcine gelatin and 0.5% TritonX-100 in TBS, pH 7.2), and incubated overnight at 4°C with the following primary antibodies dissolved in SUMI solution: rabbit anti-pACCα-Ser79 (PA5-17725, Invitrogen-Thermofisher; Carlsbad, CA, USA), goat anti-GFAP (SAB2500462; Sigma-Aldrich; Saint Louis, MO, USA), and goat anti-Iba1 (ab107519, Abcam; Cambridge, UK). Brain sections were washed in TBS and incubated with the respective secondary antibodies: donkey anti-rabbit Alexa647 (A21206, Invitrogen-Thermofisher; Carlsbad, CA, USA) and donkey anti-goat Alexa488 (A21206, Invitrogen-Thermofisher; Carlsbad, CA, USA) diluted in SUMI for 2 h at room temperature. Sections were washed in TBS and incubated with DAPI (D3571, Life Technologies; Carlsbad, CA, USA) and / or NeuroTrace™ 500 / 525 (N21480, Invitrogen-Thermofisher; Carlsbad, CA, USA) dissolved in TBS. Images were acquired as z-stacks using a confocal microscope (TCS SP8 Leica; Wetzlar, Germany) with a 20x objective immersed in glycerol and a step size of 3 μm in the z-direction. Acquired images were processed using ImageJ / FIJI. Quantification was based on visualization of cell bodies using pACCα-Ser79 staining (PA5-17725, Invitrogen-Thermofisher; Carlsbad, CA, USA), which showed predominantly neuronal profiling.The presence or absence of pACCα-Ser79 in neurons within the VMH was assessed by using co-staining with Neurotrace500 / 525 (N21480, Invitrogen-Thermofisher; Carlsbad, CA, USA). Distinct cells staining for pACCα-Ser79 were not discarded as part of the quantification.

[0141] Dual SF1 and pACCα-Ser79 immunofluorescence staining Mice were deeply anesthetized and perfused transcardially with 0.9% saline followed by 4% PFA. Brains were removed, post-fixed overnight in 4% PFA at 4°C, washed with ice-cold 0.1 M PBS to remove excess fixative solution, and then transferred to 30% sucrose in 0.1 M PBS (pH 7.4) overnight at 4°C, followed by freezing at -80°C. Sections 20 μm thick were obtained with a cryostat. Sections centered on the VMH were selected (-1.34 mm to -1.94 mm from bregma) and processed for double immunofluorescence staining of SF1 neurons and pACCα-Ser79.

[0142] Briefly, brain sections were washed three times for 5 min with 0.1 M PBS and then incubated with blocking solution (5% normal donkey serum in 0.1 M PBS containing 0.3% TritonX-100) for 2 h at room temperature. Next, sections were washed twice for 5 min with 0.1 M PBS and then incubated with the first primary antibody (SF1, 1:200 diluted in 0.1 M PBS; ab65815, Abcam; Cambridge, UK) overnight at 4°C. After three washes for 5 min with 0.1 M PBS, sections were incubated with donkey anti-rabbit Alexa Fluor594 (1:1000; A21207, Life Technologies) in 0.1 M PBS for 2 h at room temperature.

[0143] Since the two primary antibodies were obtained from the same species (i.e., rabbit), to avoid cross-reactivity between both anti-rabbit primary antibodies, brain sections were incubated with AffiniPure Fab fragment goat anti-rabbit IgG (1:40; 111-007-003, Jackson ImmunoResearch; West Grove, PA, USA) at room temperature for 4 h to saturate the remaining open binding sites on the first primary antibody. To determine the saturation concentration of the Fab fragment antibody, we performed a titration curve to determine the optimal concentration of the Fab fragment antibody. After extensive washing, sections were incubated with the second primary antibody (pACCα-Ser79, 1:100 dilution in 0.1 M PBS; PA5-17725, Invitrogen) overnight at 4°C. Sections were then washed three times for 5 min in 0.1 M PBS before being incubated with donkey anti-rabbit Alexa488 (1:1000; 711-545-512, Jackson ImmunoResearch) for 2 h at RT.

[0144] After a final step consisting of three 0.1 M PBS washes, sections were mounted using Vectashield containing DAPI (Vector Laboratories; Peterborough UK) and stored in the dark at 4°C until imaging.

[0145] Images were acquired using a confocal Leica TCS SP-5-X microscope equipped with an oil immersion 63x objective, using a 3x zoom. All pictures were taken using the exact same microscope settings (laser power and gain) to ensure similar imaging conditions for all pictures of VMH sections (n=4 animals per group). Images were imported into Fiji (NIH; Bethesda, MD, USA) where maximum intensity projections were made and brightness and contrast were adjusted equally. For quantification, each SF1 positive neuron per section was manually selected and the intensity of the pACC signal was analyzed and expressed as a percentage relative to the control. Additionally, pACC levels in other hypothalamic nuclei (ARC, DMH and PVH) were quantified as negative controls for sEV injection.

[0146] Western blotting For their characterization, 10 μg of sEVs were separated by PAGE. After migration, proteins were transferred to nitrocellulose membranes and incubated with the following antibodies: Alix (Biolegend; San Diego, CA, USA), CD9 (BD Pharmingen; San Diego, CA, USA), CD81, TSG101, and GRP94 (Santa Cruz Biotechnology, Dallas, TX, USA).

[0147] II cells with Lamp2b-RVG and sEVs, purified sEVs (native or Lamp2b-RVG modified) and their translocation to the membrane were immunoblotted with Lamp2b (Abcam; Cambridge, UK) following the same Western blotting protocol as above.

[0148] As previously shown 12、13、15~18、44 Dissected VMH or peripheral tissues were homogenized and lysed in a buffer containing 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% TritonX-100 vol / vol, 0.1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, and a protease inhibitor cocktail, as previously described. 12、13、15~18、44 Protein lysates were subjected to SDS-PAGE, electrophoretically transferred to PVDF membranes and probed with the following antibodies: pACCα-Ser79, ACCα (Cell Signaling, Danvers, MA, USA), UCP1, UCP3, Lamp2b (Abcam; Cambridge, UK), PGC1α, PGC1β, GRP94 (Santa Cruz Biotechnology, Dallas, TX, USA), β-actin, α-tubulin (Sigma-Aldrich; St. Louis, MO, USA) and GAPDH (Merck Millipore; Billerica, MA, USA).

[0149] Each membrane was then incubated with the corresponding secondary antibody: anti-mouse or anti-rabbit (DAKO; Glostrup, Denmark). Values ​​were expressed relative to the protein levels of α-tubulin (for BAT, skeletal muscle and heart) or β-actin (for the rest of the analyzed tissues). For sEVs, values ​​were expressed relative to the total protein content measured by Ponceau S. Autoradiographic films were scanned and band signals quantified by densitometry using ImageJ-1.33 software (NIH; Bethesda, MD, USA). Representative images of all proteins are shown, although in the case of loading controls, each protein was corrected by its own internal control (β-actin or α-tubulin), as explained above, and representative gels are displayed. In all figures showing images of gels, all bands in each picture always come from the same gel, although they may have been spliced ​​for clarity and are marked with vertical lines.

[0150] statistical analysis Data are expressed as mean ± SEM and, when data are relativized, they are given as percentages of the appropriate control. Statistical significance was determined by two-tailed (at least one-tailed) Student's t-test (when two groups were compared) or two-tailed ANOVA (when more than two groups were compared) followed by post-hoc Bonferroni test. Relationships between continuous variables were analyzed by simple correlation (Pearson's test). P<0.05 was considered significant.

[0151] Data availability The data supporting the findings of this study are available from the corresponding authors upon reasonable request.

[0152] result Generation and characterization of neuron-targeting dendritic cell-derived sEVs As is required for any organic delivery system, immunologically inert vesicles had to be designed to limit the host immune response. 27、28Immature dendritic cells were used to generate large amounts of sEVs with low expression of T cell activating factors such as major histocompatibility complex II (MHC-II) and cluster of differentiation 80 and 86 (CD80 and CD86). To confer neural targeting ability to the produced sEVs, immature dendritic cells were genetically modified to transiently express a fusion protein, as previously reported, consisting of (i) lysosomal-associated membrane protein 2b (Lamp2b), a protein highly expressed in the sEV membrane, and (ii) fused to a specific glycoprotein derived from the neurotrophic rabies virus (RVG), which allows crossing of the blood-brain barrier (BBB) ​​via binding to the nicotinic acetylcholine receptor (nAChR). Three days after transfection with the Lamp2b-RVG plasmid, sEVs were isolated, purified, and analyzed. Neuron-targeted Lamp2b-RVG sEVs showed higher levels of Lamp2b expression compared to native ones (Figure 9a-b), suggesting good integration of Lamp2b-RVG in the sEV membrane. Interestingly, using this strategy, it was demonstrated that RVG localized to the outer membrane of sEVs without affecting their physical properties. 28 In agreement with the literature, Lamp2b-RVG sEVs had a size distribution of 30-150 nm, as determined by nanoparticle tracking analysis (NTA, camera level 9, shutter: 607, and gain: 15, Figure 1a). These results were confirmed by electron microscopy analysis (Figure 1b). The sizes of sEVs obtained by the two different methods (NTA and electron microscopy) differ slightly. This size difference is explained by the fact that the procedure of staining sEVs for electron microscopy induces their dehydration, reducing their size. 31 sEVs expressed specific markers such as ALIX, TSG101, CD9, and CD81 (Fig. 1c), and lacked GRP94, a marker commonly used to assess EV purity (Fig. ​(Fig.9c).

[0153] The ability of sEVs to efficiently deliver nucleic acids was assessed in vitro using fluorescent nucleic acid probes, siRNA labeled with Texas Red, and a plasmid encoding green fluorescent protein (GFP). Once sEVs were exogenously loaded with nucleic acids, they were incubated with immature dendritic cells for 2, 6, or 24 h, and the respective cellular fluorescence was analyzed using confocal microscopy. siRNA-Texas Red-loaded sEVs induced red fluorescence of immature dendritic cells after 2 h compared to control conditions (non-loaded sEVs, Figure 1d). Similarly, GFP plasmid-loaded sEVs induced green fluorescence of immature dendritic target cells after 6 and 24 h compared to control conditions (Figure 1e).

[0154] We next assessed the ability of Lamp2b-RVG sEVs to cross the BBB after intravenous injection. sEVs were labeled with the near-infrared dye DID, which fluoresces when incorporated into lipid structures. To avoid nonspecific residual fluorescence of DID, the labeled sEVs were washed twice before injection. DID-labeled native (control) and DID-labeled Lamp2b-RVG sEVs were subsequently injected into the tail vein of nude mice. Anesthetized live mice were imaged using the DID-fluorescence spectrum (excitation maximum: 644 nm; emission maximum: 665 nm) at 30 min, 2, 4 and 6 h using the in vivo fluorescence imaging system MAESTRO. The level of resolution from the fluorescent whole-mouse imaging (Fig. 1f) did not allow us to precisely determine from which tissue the DID-fluorescence signal originated. Therefore, animals were sacrificed at 6 h, the different organs were harvested and the fluorescence was analyzed ex vivo. Interestingly, the two populations of sEVs, native (control) and Lamp2b-RVG, were mostly distributed in the lung, spleen and liver, with lower lung targeting for the Lamp2b-RVG population compared to that of the control (Figure 1g).

[0155] This distribution profile is consistent as these organs are highly vascularized and involved in detoxification processes. Interestingly, Lamp2b-RVG sEVs showed significantly increased localization in nAChR-expressing tissues, such as the heart (Fig. 1g) and brain (Fig. 1h-i), compared to the native sEV control condition.

[0156] The percentage of brain uptake of total fluorescence was 2.3 ± 0.3%, and when fluorescence was corrected for tissue weight, the percentage of uptake increased to 5.3 ± 0.7% fluorescence / mg. Furthermore, DID-specific fluorescence in the brain was significantly increased using the Lamp2b-RVG targeting strategy (control: 100 ± 5.01; Lamp2b-RVG: 129 ± 0.65; P < 0.01; Figure 1h-i), confirming its suitability for increasing targeting of sEVs to the central nervous system (CNS).

[0157] Having demonstrated that sEVs could reach the brain, the next step was to restrict the specificity of delivery to SF1 cells, a unique cell population that expresses this factor exclusively in the VMH and specifically in the CNS 32 , 33 .

[0158] For this, we designed a plasmid encoding the AMPKα1-DN mutant expressed under the control of the SF1 promoter (SF1-AMPKα1-DN) (Figure 9d). Purified neuron-targeted Lamp2b-RVG sEVs were subsequently loaded with SF1-AMPKα1-DN. Interestingly, loading did not alter either the morphological characteristics or the size of the sEVs, as measured by NTA at camera level 9 (shutter: 607, gain: 15) and electron microscopy (Figures 1a and 9e-f). Furthermore, the amount of encapsulated SF1-AMPKα1-DN plasmid was assayed by TritonX-100 0.2% (Figures 9g-h) with or without lysis of the sEVs, indicating the presence of the SF1-AMPKα1-DN plasmid in the membrane and inside the sEV core.

[0159] We then evaluated the efficacy of this strategy by assaying the phosphorylation level of acetyl-CoA carboxylase α (pACCα), a major downstream target of AMPK, in the hypothalamic cell line GT1-7, which demonstrated that SF1 34 Endogenously expressing and possessing numerous neuronal properties 35 , making it an excellent model for preliminary AMPK neuron research. 36、37 The data showed that when GT1-7 cells were incubated with SF1-AMPKα1-DN loaded sEVs for 24 h, the phosphorylation level of ACCα was significantly decreased compared to the non-loaded control sEVs (Figure 1j-k). Of note, no changes in pACC levels were found when other CNS-derived cells that do not express SF1 (e.g., primary astrocytes and Neuro2A cells) were incubated with SF1-AMPKα1-DN loaded sEVs (Figure 9i-j). Taken together, these data indicate that the generated Lamp2b-RVG sEVs loaded with SF1-AMPKα1-DN mutants (i) are uniform in size and morphology, (ii) can deliver nucleic acids, (iii) target the brain after intravenous injection, and (iv) specifically regulate AMPKα1 activity under the control of the SF1 promoter in vitro.

[0160] Central treatment with SF1-AMPKα1-DN-loaded sEVs induced feeding-independent weight loss in obese mice First, we evaluated the efficacy of stereotactic central delivery of SF1-AMPKα1-DN loaded sEVs in the VMH of diet-induced obese (DIO) mice fed a 60% high-fat diet (HFD). The results demonstrated that SF1-AMPKα1-DN sEVs induced feeding-independent weight loss for 3 days when administered into this nucleus (Figure 2a-b). Next, we used the results of the stereotactic central delivery of SF1-AMPKα1-DN sEVs, as previously shown. 15~17We assayed the levels of pACCα (a surrogate marker for AMPK activity) in microdissected hypothalamic extracts. The specificity of the micropunch was verified by measuring the mRNA levels of Sf1 (a specific marker for the VMH), proopiomelanocortin (Pomc; a specific marker for the hypothalamic arcuate nucleus, ARC) and hypocretin / orexin (Hcrt; a specific marker for the lateral hypothalamic area, LHA), respectively (Figure 10). Our data showed that SF1-AMPKα1-DN-loaded sEVs induced a significant decrease in phosphorylation levels of ACCα in the VMH, but not in the ARC or LHA (Figures 2c-d). This was associated with increased BAT thermogenesis, as indicated by increased temperature in the interscapular region and elevated BAT uncoupling protein 1 (UCP1) protein levels (Figures 2e-h). Overall, these data recapitulate the effects of viral gene-mediated treatment with the AMPKα1-DN isoform as well as the phenotype of SF1 AMPK null mice. 12~18 .

[0161] Systemic treatment with SF1-AMPKα1-DN-loaded sEVs modulates hypothalamic neuronal AMPK activity Next, we aimed to investigate the ability of systemic administration of SF1-AMPKα1-DN loaded sEVs in regulating AMPK activity in the hypothalamus of DIO mice. First, we evaluated the efficiency of our treatment by assaying the expression of SF1-AMPKα1-DN transgene in several tissues 24 h after intravenous injection of loaded sEVs. The transgene was detected only in VMH samples, but not in any of the other evaluated organs, including SF1-expressing (i.e., adrenal gland, pituitary gland, and testis) or non-SF1-expressing (i.e., BAT, liver, skeletal muscle, and heart) organs (Fig. 3a). Overall, this data indicates that expression of the AMPKα1-DN transgene is essential for the regulation of AMPK activity in neuronal cells (given RVG-dependent tropism), including SF1-expressing neurons of the VMH (considering SF1-driven expression). 28、30), demonstrating the specificity of our strategy. To confirm these results, we assayed the phosphorylation levels of ACCα in several tissues after intravenous injection. SF1-AMPKα1-DN loaded sEVs induced a significant decrease in the phosphorylation levels of ACCα and AMPK activity in the VMH (Fig. 3b-d). In this context, SF1-AMPKα1-DN sEV-treated mice showed a significant decrease in the number of pACCα Ser79-positive neurons in the VMH compared to controls, as demonstrated by colocalization of pACCα and Neurotrace (Fig. 3e-f). We also assessed the presence of pACCα in non-neuronal cells by co-staining for glial fibrillary acidic protein (GFAP, an astrocyte marker) and ionized calcium-binding adaptor molecule 1 (Iba1, a microglial marker) in the VMH. However, our analysis did not detect pACCα colocalization with GFAP- or Iba1-expressing cells (Figure 11a-b). To gain more insight into the specificity of our treatment, we analyzed the levels of pACCα-Ser79 in SF1 neurons of the VMH by double immunofluorescence assay. Our data showed that pACC immunoreactivity was significantly reduced in SF1 neurons of mice treated with SF1-AMPKα1-DN sEVs when compared to negative controls (Figure 3g-h), demonstrating the specificity of this technique (Figure 11c).

[0162] Notably, pACCα levels were not decreased in adjacent hypothalamic nuclei such as the ARC, dorsomedial (DMH), and paraventricular (PVH) (FIG. 11d).

[0163] Overall, this evidence demonstrates that expression of the SF1-AMPKα1-DN transgene occurs in SF1 neurons within the VMH but not in other hypothalamic cell populations.

[0164] To further confirm the specificity of our treatment with SF1-AMPKα1-DN loaded sEVs, we examined pACC levels in other parts of the CNS and peripheral tissues. No changes were found in phosphorylation levels of ACCα in any other brain regions tested (e.g., cortex, thalamus and cerebellum; Fig. 12a) or peripheral tissues such as liver, adrenal gland, testis, BAT, heart and skeletal muscle (Fig. 12b). SF1-AMPKα1-DN loaded sEVs did not induce changes in pACCα levels in primary brown adipocytes (Fig. 12c). These data also suggest that potential side effects to blunting AMPK signaling in other tissues (including those expressing SF1, such as testis and adrenal gland) are likely negligible. However, the use of assays relying on total protein extracts in peripheral tissues where SF1 cells are scarce (e.g., limited to testicular Leydig cells) may be of concern. 32、33 , may not be considered reliable. To overcome this limitation and further evaluate the possible effects of the treatment on testicular and adrenal function, we analyzed circulating levels of testosterone and corticosterone (CORT) and mRNA expression of key steroidogenic enzymes in the testes and adrenal glands of control and SF1-AMPKα1-DN-loaded sEV-treated mice. The data showed that treatment with SF1-AMPKα1-DN-loaded sEV did not induce significant changes in either circulating testosterone levels (Figure 12d) or the mRNA levels of several enzymes involved in testicular steroidogenesis, such as steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (P450ssc), and 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) (Figure 12e). On the other hand, adrenal function was not affected by treatment with SF1-AMPKα1-DN loaded sEVs, as no changes in circulating CORT (Fig. S1F) or adrenal levels of P450ssc or STAR mRNA (Fig. S1G) were detected. Similarly, no changes were observed in either circulating levels of luteinizing hormone (LH; Fig. S1H) or mRNA levels of the LHβ subunit (Fig. S1I), whose pituitary production is known to be regulated by SF1. 38 .

[0165] Systemic treatment with SF1-AMPKα1-DN-loaded sEVs induced feeding-independent weight loss in obese mice To evaluate the efficiency of SF1-AMPKα1-DN-loaded sEVs in regulating body weight, DIO mice were used. Notably, they were injected systemically into the tail vein of mice to avoid any procedures / surgeries involving direct administration to the CNS.

[0166] First, we evaluated how long the SF1-AMPKα1-DN transgene was expressed in VMH SF1 neurons after peripheral treatment with loaded sEVs. Our data showed that the AMPKα1-DN transgene could be detected in the VMH for 24 h (Fig. 3i-j). However, keeping with experiments involving only central administration (Fig. 2h), an increase in BAT UCP1 expression could be detected up to 48 h after intravenous injection (Fig. 12j). For these reasons, we chose a strategy based on one injection every 3 days for 6 days. We found that after 6 days of treatment (injections on days 0 and 3), intravenous injection of SF1-AMPKα1-DN loaded sEVs induced a significant and marked feeding-independent weight loss in DIO mice when compared to control sEVs (Fig. 4a-d), accompanied by a concomitant increase in EE (Fig. 4e). However, respiratory quotient (RQ) and locomotor activity (LA) were not modified (Figures 4f-g). Notably, sEV-induced weight loss was associated with a decrease in adiposity, as demonstrated by nuclear magnetic resonance analysis (Figures 4h-k).

[0167] Next, we investigated the long-term effects of sEV treatment in DIO mice (administration every 3 days for 4 weeks). The results showed that SF1-AMPKα1-DN loaded sEV-injected DIO mice showed a significant long-term reduction in body weight (Figure 4l-n) with no change in food intake (Figure 4o-p) when compared with mice treated with control sEV, which normally gained weight. Of note, the effect of sEV persisted when treatment was stopped. Indeed, DIO mice treated with SF1-AMPKα1-DN loaded sEV did not show weight recovery until 2 weeks after injections were stopped (washout) when compared with mice treated with control sEV (Figure 4l-m). Overall, these data indicate that the weight loss induced by SF1-AMPKα1-DN loaded sEV is not transient and the washout period does not imply a rebound effect sufficient to catch up with the weight of the control group. The weight-reducing effect of sEVs was associated with a trend to reduce circulating leptin levels (Fig. S13a), whereas no changes in growth / differentiation factor 15 (GDF15; Fig. S13b) were observed.

[0168] Assessment of circulating inflammatory markers showed no changes in interleukin-6 (IL-6) (Fig. 13c) and interferon gamma-inducible protein 10 (IP-10) levels (Fig. 13d), suggesting that sEV administration did not induce a systemic inflammatory response. The effect of SF1-loaded sEVs on circulating metabolic parameters was also evaluated. Our data showed significantly reduced non-esterified fatty acid (NEFA) circulating levels in the loaded sEV-treated group, which is consistent with increased BAT thermogenesis (see below), and no changes in total triglycerides or cholesterol (Fig. 13e-g). Finally, to assess possible adverse effects of our treatment, we analyzed the effect of SF1-AMPKα1-DN-loaded sEVs on aspartate transaminase (AST) and alanine transaminase (ALT), but since neither of them were altered (Fig. 13h-i), we ruled out a hepatic effect of sEVs. No changes were observed in any of the key cardiovascular parameters, i.e., heart rate (Figure S13j) or arterial pressure (systolic, diastolic and mean; Figure S13k-Figure S13m). Rather, there was a trend toward lower blood pressure in mice treated with SF1-AMPKα1-DN sEVs (although this was not significant), consistent with the weight loss induced by this treatment. These data indicate the lack of cardiovascular side effects of AMPK-sEVs when administered systemically.

[0169] Finally, to gain more insight into the time-dependent effects of SF1-AMPKα1-DN loaded sEV treatment, we performed a crossover study. In this new experimental setting, animals were injected into the tail vein according to a two-cycle protocol with an intercalated non-treatment period. Thus, once the functional effects of the first injection were no longer observed, a new injection was performed to evaluate the efficacy of the new treatment. The data showed that both injection cycles induced the expected feeding-independent weight loss (Figure 4q-Figure 4s).

[0170] Systemic treatment with SF1-AMPKα1-DN-loaded sEVs increased BAT thermogenesis in obese mice We next investigated whether the feeding-independent weight loss observed in DIO mice treated with SF1-AMPKα1-DN-loaded sEVs could be related to elevated BAT thermogenesis. This was justified by previous evidence indicating that genetic inhibition or ablation of AMPKα1 in these hypothalamic cells promotes brown fat activity12-18,23 and by data from our stereotaxic (Figure 2e-h) and time-response (Figure 12j) experiments. Mice treated with SF1-AMPKα1-DN-loaded sEVs showed higher BAT temperatures starting from day 1 post-injection, which persisted throughout the treatment (Figure 5a-c).

[0171] The evidence presented suggested that peripheral treatment with SF1-AMPKα1-DN loaded sEVs induced BAT thermogenesis, but not weight loss associated with feeding. Therefore, before performing mechanistic experiments involving modulation of BAT function, we aimed to address possible correlations between these variables. Our data revealed a highly significant negative correlation between weight change and BAT temperature (P<0.0001): mice administered SF1-AMPKα1-DN loaded sEVs were the ones that lost the most weight and had higher BAT temperatures (Fig. 5d). Notably, food intake was similar in both groups and no association was found (Fig. 5e). Overall, this evidence suggested that increased BAT function led to increased EE, explaining the weight loss effect of this sEV strategy. To gain further insight into the thermogenic effect of these sEVs targeting hypothalamic AMPK, we analyzed BAT temperature in a crossover experiment (Fig. 4q-4s). Notably, when treatment was discontinued, SF1-AMPKα1-DN-loaded sEV-induced BAT temperature reverted to control / basal values ​​(Figures 5f-h), indicating that the observed effects were time- and treatment-dependent.

[0172] Furthermore, we investigated their effects on tail base temperature, a well-known thermoregulatory mechanism in rodents. 39 The loaded sEVs induced a slight but non-significant increase in tail base temperature (Figure 5i-j), indicating a trend towards heat dissipation through the tail.

[0173] Systemic treatment with SF1-AMPKα1-DN-loaded sEVs induced the BAT thermogenic program and glucose uptake BAT of SF1-AMPKα1-DN-loaded sEV-treated DIO mice showed increased protein levels of key thermogenic markers, such as UCP1, uncoupling protein 3 (UCP3), and peroxisome proliferator-activated receptor gamma coactivator 1α and β (PGC1α and PGC1β) (Figures 6a-b at 6 days and 14a at 28 days). Consistent with these data, injection of SF1-AMPKα1-DN-loaded sEVs induced higher BAT 18F-FDG uptake when compared to liver (used as control tissue) (Figures 6c-d), indicating higher BAT activation. Interestingly, injection of SF1-AMPKα1-DN-loaded sEVs was also associated with a non-significant trend to increase browning of subcutaneous white adipose tissue (scWAT), as suggested by slightly increased UCP1 staining (Figures 6e-f).

[0174] Overall, this evidence indicates that systemic administration of SF1-AMPKα1-DN-loaded sEVs targeting hypothalamic SF1 neurons induces changes in BAT activity. Of note, this effect is likely not related to a nonspecific effect of sEVs on brown adipocytes (when SF1 is not expressed) for two main reasons: (i) inhibition of BAT AMPKα1 has recently been reported to cause dysfunction rather than activation in this tissue,40 and (ii) importantly, no changes in AMPK signaling were found in BAT, either after sEV treatment in vivo (Fig. S12b) or when sEVs were fed to primary BAT adipocytes in vitro (Fig. S12c), excluding the presence of confounding nonspecific effects. Furthermore, no changes were found in the skeletal muscle thermogenic program (Fig. S14b), indicating that the increase in EE observed upon systemic administration of sEVs is driven by BAT and not muscle thermogenesis.

[0175] Systemic treatment with SF1-AMPKα1-DN-loaded sEV-induced BAT induced thermogenesis and weight loss via activation of the SNS BAT thermogenesis is primarily controlled by the SNS via β3-adrenergic receptors (β3-ARs). Therefore, we investigated whether regulation of BAT following systemic injection of sEVs targeting AMPKα1 in VMH-SF1 neurons is mediated by the SNS. SF1-AMPKα1-DN-loaded sEVs increased total BAT sympathetic nerve traffic, which was directly recorded by microneurography (Figure 7a-b). Transection of the BAT nerve distal to the recording site allowed for the measurement of efferent sympathetic nerve activity. Mice treated with SF1-AMPKα1-DN-loaded sEVs showed significantly elevated efferent BAT sympathetic nerve activity (SNA) compared to controls. However, calculated afferent BAT nerve activity did not differ between the two groups. These data demonstrate that SF1-AMPKα1-DN-loaded sEVs stimulated efferent, but not afferent, sympathetic outflow, which is consistent with a centrally mediated effect of the treatment. Consistent with the SNA data, pharmacological inhibition of the β3-AR by subcutaneous administration of the specific β3-AR antagonist, SR59230A 12、13、15~18、44 prevented the loss of body weight induced by peripheral intravenous injection of SF1-AMPKα1-DN-loaded sEVs without impeding feeding (Figure 7c-d).

[0176] Consistent with the increased body weight following β3-AR blockade, treatment with SR59230A abolished the increase in BAT temperature (Figure 7e-f) and UCP1 protein levels (Figure 7g-h). Notably, when administered alone, SR59230A did not promote changes in any of the parameters analyzed (Control+Vehicle vs. Control+SR59230A: (i) Body weight: -1.27±0.09 vs -1.30±0.42, not significant; (ii) Food intake: 2.49±0.07 vs 2.58±0.17, not significant; (iii) BAT temperature: 36.8±0.18 vs 36.7±0.07, not significant; (iv) UCP1 BAT: 100±8.2 vs 84.1±8.3, not significant). Overall, this evidence indicates that systemic injection of SF1-AMPKα1-DN-loaded sEVs promotes weight loss acting on SF1 neurons in the VMH, independent of feeding, and leads to increased BAT thermogenesis via SNS-induced β3-AR activation. Notably, the fact that no changes were observed in key cardiovascular parameters, as shown (Figure S1c–1d), suggested that the sympathetic stimulation exerted by sEVs is specific to BAT.

[0177] Systemic treatment with SF1-AMPKα1-DN-loaded sEVs induced BAT thermogenesis and weight loss in thermoneutral conditions We hypothesized that in a non-thermonutral environment (22–23 °C for housing mice), basal activation of BAT may mask the effect of sEVs. 41、45 Therefore, we aimed to investigate whether the effect of SF1-AMPKα1-DN loaded sEVs depends on the ambient temperature. However, the data showed that treatment with SF1-AMPKα1-DN loaded sEVs in DIO mice housed in thermoneutral conditions (30 °C) induced a significant weight loss (Figures 8a-b), independent of feeding (Figures 8c-d), and was associated with increased BAT thermogenesis (Figures 8e-f) and increased BAT UCP1 protein content (Figures 8g-h). Overall, this evidence demonstrates that SF1-AMPKα1-DN loaded sEVs regulate energy balance and body weight by targeting BAT thermogenesis.

[0178] UCP1 is essential for the thermogenic and weight loss effects induced by systemic SF1-AMPKα1-DN-loaded sEVs Finally, we investigated whether the effect of SF1-AMPKα1-DN sEVs on BAT thermogenesis and body weight was dependent on UCP1 expression. SF1-AMPKα1-DN sEVs induced feeding-independent but thermogenesis-dependent weight loss in wild-type mice (Fig. 8i, 8k, 8m, and 8n), but this effect was completely absent in ucp1 null mice (Fig. 8j, 8l, 8o, and 8p). Overall, these data demonstrate that UCP1 plays an essential role in mediating the central effect of SF1-AMPKα1-DN sEVs on thermogenesis and energy balance, while simultaneously confirming that contributions from other peripheral tissues such as muscle are negligible.

[0179] Consideration The development of strategies to halt the current obesity pandemic has been hindered primarily due to (i) the inherent redundancy of homeostatic mechanisms regulating body weight, (ii) their resilience to homeostatic perturbations as a result of counterregulatory responses (i.e., reduced feeding leads to reduced EE), (iii) the limited specificity of most drugs available so far, and (iv) deleterious side effects. 1~5 It would therefore be interesting to generate new genetic strategies / resources that allow for more precise targeting and thus greater specificity.

[0180] Due to their composition, sEVs can be used as drug shuttles and therefore can be used to target molecules to specific cells. 19~22 , and thus can be used for prognosis, biomarkers and innovative treatments. 19~22、24~26 Indeed, their properties, such as biocompatibility and low immunogenicity, make them ideal for reaching the CNS. 27、28To target central mechanisms regulating energy balance, crossing the BBB is a major challenge in delivering agents of interest. To circumvent this limitation, engineered sEVs expressing the RVG peptide fused to Lamp2b on their surface have been developed, allowing for specific neuronal targeting. 28 , not specific to one neuronal population of any given brain region. We have utilized this approach using sEVs as cargo for DNA plasmids of interest in an obesity-driven context. Thus, in this study, we have demonstrated that sEVs mediate the expression of central and canonical pathways that regulate energy balance, namely hypothalamic AMPK. 3、8、11 developed sEVs as a delivery tool to specifically target SF1 neurons in the VMH.

[0181] In particular, AMPK action shows high anatomical and isoform-dependent specificity, and anorexia is induced by selective loss of the AMPKα2 isoform in agouti-related protein (AgRP) neurons in the hypothalamic arcuate nucleus (ARC). 46 Inhibition of AMPKα1 activity in SF1 neurons of the VMH increases energy expenditure by stimulating SNS-driven BAT thermogenesis 17、18 Of note, mice with selective loss of AMPKα1 in SF1 neurons were resistant to DIO18, suggesting that targeting this isoform in hypothalamic populations could be an interesting target against obesity. However, implementation of this strategy requires a high level of hypothalamic specificity, as any side effects associated with peripheral inhibition of AMPK would have the opposite effect, exacerbation of insulin resistance and diabetes. 3、47、48, thus enhancing the importance of treatment specificity and safety. Therefore, we developed sEVs exogenously loaded with a plasmid encoding the AMPKα1-DN mutant under the control of the SF1 promoter. Notably, these sEVs were administered peripherally in the tail vein to avoid any central / brain manipulation and to render them acceptable for potential therapeutic use. Remarkably, the data showed that SF1-AMPKα1-DN-loaded sEVs promoted a remarkable feeding-independent weight loss effect due to an increase in SNS-mediated UCP1-dependent BAT thermogenesis (as demonstrated by the lack of effect in ucp1 null mice) and was not associated with either a systemic inflammatory response or hepatic and cardiovascular side effects. Notably, the fact that this effect occurs in the absence of appetite compensatory changes in SF1-AMPKα1-DN-loaded sEV-injected mice has translational relevance, since it eliminates the undesirable rebound effects that typically characterize dietary interventions, as also demonstrated by our long-term and crossover treatments. 5 .

[0182] Notably, as demonstrated by our colocalization analysis, SF1-AMPKα1-DN loaded sEVs, despite reaching peripheral tissues, only regulated AMPK signaling in the VMH, specifically in SF1 neurons. This is attributed to (i) RVG-mediated tropism to neurons28 and (ii) SF1-driven expression of the AMPKα1-DN transgene. Notably, no detectable changes in AMPK activity were found in (i) other hypothalamic adjacent nuclei, (ii) peripheral SF1-expressing tissues such as testis, adrenal gland and pituitary gland, and (iii) peripheral non-expressing SF1 tissues such as liver, BAT, heart and skeletal muscle. Overall, these data indicate that systemic treatment with our developed SF1-AMPKα1-DN loaded sEVs was able to induce weight loss by specifically inhibiting AMPKα1 function in SF1 neurons of the VMH. This demonstrates that the hypothalamic network can be selectively targeted by peripherally delivered agents opening new therapeutic possibilities for obesity and other neurological disorders. Strengthening this idea, our data also demonstrated the absence of hepatic and cardiovascular side effects of AMPK-sEVs when administered systemically, indicating that targeting hypothalamic AMPK may bypass some of the secondary effects associated with treatments acting on peripheral mechanisms regulating energy balance and metabolism. 5 .

[0183] Strategies for the development of successful treatments for obesity have been focused mainly on peripheral approaches, given the inherent complexity of targeting the brain. However, increasing knowledge of the hypothalamic mechanisms controlling energy homeostasis has revealed that specific modulation of neural circuits in distinct regions may provide novel and more effective targets for drug development. Interestingly, many of the key players in energy balance that have been the basis for the development of new treatments for obesity (leptin, ghrelin, glucagon-like peptide-1 agonists, glucagon, etc.) 1~6 , likely acting via hypothalamic AMPK 3 .

[0184] However, targeting specific neurons in the CNS was considered a difficult task. Here, we provide evidence for the ability of sEVs to be used as natural biocarriers as an alternative to more traditional delivery systems in the treatment of obesity, limiting inflammatory responses, and enhancing highly selective cellular actions, namely AMPKα1 in the SF1 neuron of the VMH. Thus, sEVs with genetic tools open new avenues in the rational design of new strategies for the treatment of obesity and related comorbidities, as well as possibly other neurological disorders.

[0185] Example 2 Genetic obesity is divided into two categories: monogenic obesity, inherited in a Mendelian manner, which is typically rare, early onset and severe, and polygenic obesity, which is the result of hundreds of polymorphisms, each with a small effect. In the case of monogenic obesity, there are few treatment options. For example, if the mutation is accompanied by a lack of a ligand, such as a hormone (resulting in a hypohormonal syndrome), one possibility could be replacement therapy. That strategy has given different degrees of efficiency depending on the factors. However, if the mutation occurs on a receptor, such as the leptin receptor (LEPR) or the melanocortin 4 receptor (MC4R), treatment options are more limited and even bariatric surgery may fail.

[0186] Hypothalamic AMP-activated protein kinase (AMPK) is a canonical regulator of energy balance and metabolism at the systemic level. AMPK is a key downstream factor for both the anorexic and thermogenic actions of leptin in brown adipose tissue (BAT). We used small extracellular vesicles (exosomes) as cargo of DNA sequences to specifically inhibit hypothalamic AMPKα1 (using dominant-negative mutant AMPKα1-DN) in steroidogenic factor 1 (SF1) neurons of the ventromedial hypothalamus (VMH), a key population that regulates BAT thermogenic activity. Remarkably, when diet-induced obese (DIO) mice were treated systemically (intra-tail vein) with SF1-AMPKα1-DN sEVs, these mice showed significant feeding-independent weight loss associated with increased sympathetic activation and uncoupling protein 1 (UCP1)-dependent thermogenesis in BAT. Importantly, no metabolic, endocrine or cardiovascular inflammatory responses or other adverse effects occurred.

[0187] The objectives of this study were to use sEVs carrying SF1-AMPKα1-DN in db / db mice to address i) whether our strategy is effective in treating genetic forms of obesity, and ii) whether targeting the central mechanisms regulating BAT thermogenesis may provide a new therapeutic option for the treatment of LEPR deficiency.

[0188] 1. Materials and Methods 1.1.Animals Male null LEPR (db / db) and wild-type (WT) littermate mice (C57 / BL / 6J; 8 weeks old; Janvier Labs) were used for the experiments. They were allowed free access to water and standard laboratory chow (Scientific-Animal-Food-Engineering). Seven to eight mice / group were used. Experiments were performed in compliance with the International Law on Animal Experimentation and approved by the USC Ethics Committee (15012 / 2020 / 010).

[0189] 1.2 sEV generation, validation, and treatment To confer neuronal targeting capability to sEVs and limit host immune responses, we used immature dendritic cells genetically modified to express a fusion protein of lysosome-associated membrane protein 2b (Lamp2b, a protein highly expressed in sEV membranes) fused to a specific glycoprotein derived from the neurotrophic rabies virus (RVG), which allows blood-brain barrier (BBB) ​​crossing via binding to the nicotinic acetylcholine receptor (nAChR), as shown in the previous examples. We deeply characterized sEVs, as shown using electron microscopy, nanoparticle tracking analysis (NTA) membrane protein markers, as previously demonstrated {Milbank, 2021#56494}. sEVs were loaded with a plasmid encoding the AMPKα1-DN mutant expressed under the control of the SF1 promoter (SF1-AMPKα1-DN) to restrict their action to SF1 cells in the VMH. As indicated, 100 μg of unloaded sEVs or SF1-AMPKα1-DN-loaded sEVs were injected into the tail vein of mice every 3 days for 2 weeks {Milbank, 2021#56494}. Body weight and food intake were measured daily.

[0190] 1.3. Animal measurements BAT temperature (B335: Compact Infrared Thermal Imaging Camera; FLIR) and nuclear magnetic resonance (NRM; Whole Body Composition Analyzer; EchoMRI) were performed as described above.

[0191] 1.4 Analysis method Serum triglyceride levels, VMH acetyl-CoA carboxylase (pACCα / ACCα) and BAT UCP1 Western blotting, and BAT and white adipose tissue (WAT) UCP1 immunostaining were performed using the same kit (Spinreact), antibody [(pACCα-Ser 79and ACCα (Cell Signaling); UCP1 (Abcam); β-actin, α-tubulin (Sigma-Aldrich), and were performed as described using the reagents listed above. In the Western blot images, all samples were loaded on the same gel, but black lines are inserted where samples were not loaded side-by-side.

[0192] 1.5 Statistical analysis Data are expressed as MEAN±SEM. Statistical significance was determined by mixed-effects analysis (for treatment over time) or unpaired Student's t-test, with P<0.05 being significant.

[0193] 2.Results 2.1. Systemic SF1-AMPKα1-DN sEVs induce weight loss in db / db mice Intravenous injection of SF1-AMPKα1-DN sEVs promoted significant feeding-independent weight loss in wild-type (Figures 15A, 15C, 15E, and 15F) and db / db mice (Figures 15B, 15D, 15G, and 15H) and was associated with reduced adiposity (Figures 15I and 15K) and serum triglyceride levels (Figures 15J and 15L) in db / db mice.

[0194] 2.2. Systemic SF1-AMPKα1-DN sEVs increased thermogenesis and browning Inhibition of AMPKα1 in SF1 neurons of the VMH is a classical mechanism for increasing sympathetic tone on BAT, leading to increased heat production, energy expenditure, and weight loss. Our data showed that intravenous injection of SF1-AMPKα1-DN sEVs reduced pACCα levels in the VMH of both mouse models (Figures 16A and 16B), demonstrating the efficacy of our treatment in inhibiting AMPK activity in this hypothalamic nucleus.

[0195] Next, we evaluated the effect of sEV-mediated treatment on thermogenesis. Our results showed that both wild-type mice and db / db mice treated intravenously with SF1-AMPKα1-DN sEVs exhibited increased BAT temperature (FIGS. 16C-D) due to increased UCP1 protein levels (FIGS. 16E-F) and immunoreactivity (FIGS. 16G-H). Finally, our results also demonstrated that db / db mice (but not wild-type) exhibited higher UCP1 immunoreactivity in subcutaneous WAT (sWAT) and exhibited browning (FIGS. 16I-J). Overall, this evidence demonstrated that SF1-AMPKα1-DN sEV-induced weight loss in LEPR-deficient mice (and their wild-type counterparts) was associated with increased BAT thermogenesis and WAT browning.

[0196] 3. Conclusion Many of the current treatments for genetic forms of obesity are mechanistically based on replacement therapy. Such approaches are based on the use of agonists to replace the lack of ligand binding to key receptors. The search for new strategies for obesity-induced LEPR deficiency remains an unmet clinical need. Many of these patients have been unable to achieve the desired therapeutic response even when treated with new MC4R agonists.

[0197] Hypothalamic AMPK acts downstream of LEPR to regulate both feeding and BAT thermogenesis. Furthermore, we demonstrated that central targeting of AMPKα1 by an sEV-based strategy is a suitable approach for DIO in preclinical models by specifically regulating BAT thermogenesis.

[0198] With this in mind, we evaluated the efficacy of SF1-AMPKα1-DN sEV in db / db mice, a model of complete leptin resistance due to LEPR deficiency. Our results demonstrated that intravenous injection of SF1-AMPKα1-DN sEV inhibited AMPK activity in the VMH, leading to BAT thermogenesis and weight loss in wild-type and db / db mice, and also increased WAT browning. Notably, SF1-AMPKα1-DN sEV-induced weight loss was completely feeding-independent, suggesting that combination of our approach with other strategies targeting appetite (e.g., setomelanotide, or even sEVs against AMPK in other hypothalamic cell populations that regulate homeostatic and / or hedonic food intake) may allow for more significant weight loss to be achieved. This allows for a more integrated treatment of LEPR deficiency, and thus any leptin-resistant condition, by targeting both sides of the energy balance equation.

[0199] Thus, our data reveal for the first time that sEV-mediated targeting of hypothalamic AMPK, by stimulating brown fat thermogenesis and WAT browning, may be a suitable approach against this form of genetic obesity.

[0200] References 1. Clemmensen, C., Muller, TD, Finan, B., Tschop, MH & DiMarchi, R. Current and Emerging Treatment Options in Diabetes Care. Handb. Exp. Pharmacol 233, 437-459 (2016). 2. Tschop, MH, et al. Unimolecular Polypharmacy for Treatment of Diabetes and Obesity. Cell Metab 24, 51-62 (2016). 3. Lopez, M., Nogueiras, R., Tena-Sempere, M. & Dieguez, C. Hypothalamic AMPK: a canonical regulator of whole-body energy balance. Nat. Rev. Endocrinol 12, 421-432 (2016). 4. Cui, H., Lopez, M. & Rahmouni, K. The cellular and molecular bases of leptin and ghrelin resistance in obesity. Nat. Rev. Endocrinol 13, 338-351 (2017). 5. Muller, T.D., Clemmensen, C., Finan, B., Dimarchi, R.D. & Tschop, M.H. Anti-Obesity Therapy: from Rainbow Pills to Polyagonists. Pharmacol. Rev 70, 712-746 (2018). 6. Dragano, N.R.V., Ferno, J., Dieguez, C., Lopez, M. & Milbank, E. Recent Updates on Obesity Treatments: Available Drugs and Future Directions. Neuroscience (2020). 7. Kahn, B.B., Alquier, T., Carling, D. & Hardie, D.G. AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1, 15-25 (2005). 8. Schneeberger, M. & Claret, M. Recent Insights into the Role of Hypothalamic AMPK Signaling Cascade upon Metabolic Control. Front Neurosci 6, 185 (2012). 9. Hardie, D.G., Schaffer, B.E. & Brunet, A. AMPK: An Energy-Sensing Pathway with Multiple Inputs and Outputs. Trends Cell Biol (2015). 10. Carling, D. AMPK signalling in health and disease. Curr. Opin. Cell Biol 45, 31-37 (2017). 11. Lopez, M. AMPK Wars: the VMH Strikes Back, Return of the PVH. Trends Endocrinol. Metab 29, 135-137 (2018). 12. Lopez, M., et al.Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance. Nat. Med 16, 1001-1008 (2010). 13. Martinez de Morentin, P.B., et al. Nicotine induces negative energy balance through hypothalamic AMP-activated protein kinase. Diabetes 61, 807-817 (2012). 14. Whittle, A.J., et al.Bmp8b increases brown adipose tissue thermogenesis through both central and peripheral actions. Cell 149, 871-885 (2012). 15. Martinez de Morentin, P.B., et al. Estradiol regulates brown adipose tissue thermogenesis via hypothalamic AMPK. Cell Metab 20, 41-53 (2014). 16. Martins, L., et al. A Functional Link between AMPK and Orexin Mediates the Effect of BMP8B on Energy Balance. Cell Rep 16, 2231-2242 (2016). 17. Martinez-Sanchez, N., et al.Hypothalamic AMPK-ER stress-JNK1 axis mediates the central actions of thyroid hormones on energy balance. Cell Metab 26, 212-229 (2017). 18. Seoane-Collazo, P., et al.SF1-Specific AMPKalpha1 Deletion Protects Against Diet-Induced Obesity. Diabetes 67, 2213-2226 (2018). 19. Buzas, E.I., Gyorgy, B., Nagy, G., Falus, A. & Gay, S. Emerging role of extracellular vesicles in inflammatory diseases. Nat. Rev. Rheumatol 10, 356-364 (2014). 20. Milbank, E., Martinez, M.C. & Andriantsitohaina, R. Extracellular vesicles: Pharmacological modulators of the peripheral and central signals governing obesity. Pharmacol. Ther 157, 65-83 (2016). 21. Martinez, M.C. & Andriantsitohaina, R. Extracellular Vesicles in Metabolic Syndrome. Circ. Res 120, 1674-1686 (2017). 22. Malloci, M., et al.Extracellular Vesicles: Mechanisms in Human Health and Disease. Antioxid. Redox. Signal 30, 813-856 (2019). 23. Lopez, M., et al.Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin. Cell Metab 7, 389-399 (2008). 24. Tang, K., et al. Delivery of chemotherapeutic drugs in tumour cell-derived microparticles. Nat. Commun 3, 1282 (2012). 25. Dalli, J., et al.Microparticle alpha-2-macroglobulin enhances pro-resolving responses and promotes survival in sepsis. EMBO Mol. Med 6, 27-42 (2014). 26. Kamerkar, S., et al.Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546, 498-503 (2017). 27. Quah, B.J. & O'Neill, H.C. The immunogenicity of dendritic cell-derived exosomes. Blood Cells Mol. Dis 35, 94-110 (2005). 28. Alvarez-Erviti, L., et al.Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol 29, 341-345 (2011). 29. Colombo, M., Raposo, G. & Thery, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol 30, 255-289 (2014). 30. Kumar, P., et al.Transvascular delivery of small interfering RNA to the central nervous system. Nature 448, 39-43 (2007). 31. Domingos, R.F., et al.Characterizing manufactured nanoparticles in the environment: multimethod determination of particle sizes. Environ Sci Technol 43, 7277-7284 (2009). 32. Parker, K.L. & Schimmer, B.P. Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr. Rev 18, 361-377 (1997). 33. Choi, Y.H., Fujikawa, T., Lee, J., Reuter, A. & Kim, K.W. Revisiting the Ventral Medial Nucleus of the Hypothalamus: The Roles of SF-1 Neurons in Energy Homeostasis. Front Neurosci 7, 71 (2013). 34. Corley, D.R., Li, X., Lei, Z.M. & Rao, C.V. Potential regulation of GnRH gene by a steroidogenic factor-1-like protein. Mol. Hum. Reprod 6, 671-676 (2000). 35. Mellon, P.L., et al.Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5, 1-10 (1990). 36. Coyral-Castel, S., et al.The effect of AMP-activated kinase activation on gonadotrophin-releasing hormone secretion in GT1-7 cells and its potential role in hypothalamic regulation of the oestrous cyclicity in rats. J. Neuroendocrinol 20, 335-346 (2008). 37. Beall, C., et al. Mouse hypothalamic GT1-7 cells demonstrate AMPK-dependent intrinsic glucose-sensing behaviour. Diabetologia 55, 2432-2444 (2012). 38. Schimmer, B.P. & White, P.C. Minireview: steroidogenic factor 1: its roles in differentiation, development, and disease. Mol. Endocrinol 24, 1322-1337 (2010). 39. Warner, A., et al.Inappropriate heat dissipation ignites brown fat thermogenesis in mice with a mutant thyroid hormone receptor alpha1. Proc. Natl. Acad. Sci. U. S. A 110, 16241-16246 (2013). 40. Yang, Q., et al.AMPK / alpha-Ketoglutarate Axis Dynamically Mediates DNA Demethylation in the Prdm16 Promoter and Brown Adipogenesis. Cell Metab 24, 542-554 (2016). 41. Cannon, B. & Nedergaard, J. Brown adipose tissue: function and physiological significance. Physiol Rev 84, 277-359 (2004). 42. Morrison, S.F., Madden, C.J. & Tupone, D. Central neural regulation of brown adipose tissue thermogenesis and energy expenditure. Cell Metab 19, 741-756 (2014). 43. Contreras, C., et al. The brain and brown fat. Ann. Med 47, 150-168 (2015). 44. Gonzalez-Garcia, I., et al. Estradiol Regulates Energy Balance by Ameliorating Hypothalamic Ceramide-Induced ER Stress. Cell Rep 25, 413-423 (2018). 45. Alvarez-Crespo, M., et al.Essential role of UCP1 modulating the central effects of thyroid hormones on energy balance. Mol. Metab 5, 271-282 (2016). 46. Claret, M., et al. AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J. Clin. Invest 117, 2325-2336 (2007). 47. Foretz, M., Guigas, B., Bertrand, L., Pollak, M. & Viollet, B. Metformin: from mechanisms of action to therapies. Cell Metab 20, 953-966 (2014). 48. Foretz, M. & Viollet, B. Therapy: Metformin takes a new route to clinical efficacy. Nat. Rev. Endocrinol 11, 390-392 (2015). 49. Campderros, L., et al.Brown Adipocytes Secrete GDF15 in Response to Thermogenic Activation. Obesity (Silver Spring) 27, 1606-1616 (2019). 50. Johann, K., et al.Thyroid-Hormone-Induced Browning of White Adipose Tissue Does Not Contribute to Thermogenesis and Glucose Consumption. Cell Rep 27, 3385-3400 e3383 (2019). 51. Seoane-Collazo, P., et al.Central nicotine induces browning through hypothalamic kappa opioid receptor. Nat. Commun 10, 4037 (2019). 52. Johnson, D.H., Flask, C.A., Ernsberger, P.R., Wong, W.C. & Wilson, D.L. Reproducible MRI measurement of adipose tissue volumes in genetic and dietary rodent obesity models. J Magn Reson Imaging 28, 915-927 (2008). 53. Hu, H.H., Chen, J. & Shen, W. Segmentation and quantification of adipose tissue by magnetic resonance imaging. MAGMA 29, 259-276 (2016). 54. Hong, C.W., Fazeli Dehkordy, S., Hooker, J.C., Hamilton, G. & Sirlin, C.B. Fat Quantification in the Abdomen. Top Magn Reson Imaging 26, 221-227 (2017). 55. Contreras, C. & Lopez, M. Ceramide sensing in the hippocampus: The lipostatic theory and Ockham's razor. Mol. Metab 3, 90-91 (2014). 56. Heras, V., et al. Central Ceramide Signaling Mediates Obesity-Induced Precocious Puberty. Cell Metab 32, 951-966 e958 (2020). 57. Vazquez, M.J., et al. SIRT1 mediates obesity- and nutrient-dependent perturbation of pubertal timing by epigenetically controlling Kiss1 expression. Nat. Commun 9, 4194 (2018). 58. Sanchez-Garrido, M.A., et al.Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters of the Offspring: Male-Preferential Impact and Involvement of Kiss1-Mediated Pathways. Endocrinology 159, 1005-1018 (2018). 59. Zadra, G., et al. A novel direct activator of AMPK inhibits prostate cancer growth by blocking lipogenesis. EMBO Mol Med 6, 519-538 (2014). 60. Tang, Y.C., Williams, B.R., Siegel, J.J. & Amon, A. Identification of aneuploidy-selective antiproliferation compounds. Cell 144, 499-512 (2011).

Claims

1. A population of small extracellular vesicles (sEVs) comprising at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase α1 (AMPKα1-DN) mutant protein, wherein the amino acid sequence of the AMPKα1-DN mutant protein consists of SEQ ID NO: 1; the AMPKα1-DN mutant protein is operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter having at least 98% sequence identity with SEQ ID NO: 3; The sEVs are engineered to express at least one fusion protein in their outer membrane comprising a neurotropic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b. Population of small extracellular vesicles (sEVs).

2. The population of small extracellular vesicles described in claim 1, wherein the amino acid sequence of the steroidogenic factor 1 (SF1) promoter consists of SEQ ID NO:

3.

3. 3. The population of small extracellular vesicles of claim 1 or 2, wherein the fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises SEQ ID NO: 5 or a sequence having at least 90% sequence identity to SEQ ID NO:

5.

4. A population of small extracellular vesicles described in claim 1 or 2 for the treatment or prevention of obesity.

5. The population of small extracellular vesicles described in claim 4, wherein the obesity is leptin receptor (LEPR) deficiency-induced obesity.

6. A population of small extracellular vesicles as described in claim 1 or 2 for improving or reducing the rebound effect after washout of the treatment for obesity as described in claim 4.

7. 7. The population of small extracellular vesicles of claim 6, wherein the improvement or reduction in the rebound effect is measured at least 5 days after treatment washout.

8. The population of small extracellular vesicles described in claim 4, wherein the administration of the small extracellular vesicles is systemic.

9. A population of small extracellular vesicles (sEVs) that, when systemically administered, can significantly reduce the activation level of AMP-activated protein kinase (AMPK) in SF1-expressing neurons located in the ventromedial hypothalamus (VMH) compared to the activation level of AMPK in untreated SF1-expressing cells, but the reduction is not significant in other SF1-expressing tissues selected from the list consisting of the adrenal gland, the testis, or the pituitary gland; The population of small extracellular vesicles (sEVs) comprises at least one polynucleotide encoding a dominant-negative AMP-activated protein kinase α1 (AMPKα1-DN) mutant protein, wherein the amino acid sequence of the AMPKα1-DN mutant protein consists of SEQ ID NO: 1; the AMPKα1-DN mutant protein is operably linked to and under the control of a steroidogenic factor 1 (SF1) promoter having at least 98% sequence identity with SEQ ID NO: 3; The sEV is engineered to express at least one fusion protein in its outer membrane comprising a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b; The population is for the treatment of obesity via a systemic route of administration in a subject in need thereof. Population of small extracellular vesicles (sEVs).

10. The collection of claim 9, wherein the amino acid sequence of the steroidogenic factor 1 (SF1) promoter consists of SEQ ID NO:

3.

11. The collection of claim 9 or 10, wherein the nucleotide sequence of the AMPKα1-DN mutant protein consists of SEQ ID NO:

2.

12. 11. The population of claim 9 or 10, wherein the fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprises SEQ ID NO: 5 or a sequence having at least 90% sequence identity to SEQ ID NO:

5.

13. 11. The population described in claim 9 or 10, for the amelioration or reduction of the rebound effect following washout of the treatment for obesity, wherein the amelioration or reduction of the rebound effect is measured at least 5 days following washout of the treatment.

14. 11. The population of claim 9 or 10, wherein the treatment comprises reversing or ameliorating obesity.

15. 11. The population of claim 9 or 10, wherein the systemic route is an intravascular route.

16. The population of claim 1 or 2, or the population of claim 9 or 10, wherein the small extracellular vesicles have a size distribution of 30 nm to 150 nm.

17. 17. The population or population of claim 16, wherein the small extracellular vesicles further comprise one or more specific markers selected from the group consisting of ALIX, TSG101, CD9 or CD81, or any combination thereof.

18. The population of claim 16, wherein the small extracellular vesicles lack the GRP94 marker.

19. 17. The population or population for use of claim 16, wherein the small extracellular vesicles are produced or obtained from immature antigen-presenting cells characterized by having statistically significantly reduced expression of at least one T cell activation factor molecule compared to the expression of the T cell activation factor molecule in mature antigen-presenting cells.

20. 17. The population of claim 16, wherein the antigen-presenting cells are dendritic cells and the at least one T cell activator molecule is one or more T cell activator molecules selected from the group consisting of major histocompatibility complex II (MHC-II), cluster of differentiation 80 (CD80), or cluster of differentiation 86 (CD86), or a combination thereof.

21. 17. The population of claim 16, wherein the small extracellular vesicles are exosomes.

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