Cellular membrane-derived nanocarriers for brain delivery

Abstract

The present invention provides a homogeneous population of cell-derived nanoparticles, comprising (a) a surface, wherein the surface comprises a.1) a portion of an isolated cellular membrane and a.2) at least one fusion protein comprising RVG peptide fused to lysosome-associated membrane protein 2b, and (b) at least one cargo of interest.

Description

[0001] CELLULAR MEMBRANE-DERIVED NANOCARRIERS FOR BRAIN DELIVERY

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of nanobiomedicine. Particularly, the present invention relates to populations of cell-derived nanoparticles for delivery DNA specifically to the brain.

[0004] BACKGROUND ART

[0005] Brain drug delivery is one of the main challenges that nanotechnology aims to achieve. Effective drug delivery to the central nervous system (CNS) requires overcome several critical processes: (i) long blood-circulation half-lives: drugs must have prolonged circulation times to provide sufficient opportunity to enter the brain; (ii) Blood-Brain Barrier (BBB) Penetration: successful CNS drug delivery hinges on the ability to cross the BBB, a crucial step that directly affects the efficiency of drug delivery to the brain; (iii) Uptake by targeted cells: once past the BBB, drugs need to be taken up by target cells to ensure precise functioning.

[0006] Extensive efforts have been made in the design of nanocarriers to meet CNS delivery requirements. Liposomes and polymeric nanoparticles have been engineered to enhance drug loading and reduce clearance by the reticuloendothelial system (RES), thereby prolonging the blood-circulation half-lives of drugs. Furthermore, the use of natural structures such as viral vectors or extracellular vesicles (exosomes) can achieve relatively high BBB permeability.

[0007] Additionally, advancements in nanotechnology allow for the precise engineering of nanoparticles to optimize their size, shape, and surface charge. These nano drug delivery systems can be tailored with targeting ligands (proteins, antibodies, cell penetrating peptides), which interact specifically with receptors on the BBB, facilitating their transport across this barrier.

[0008] However, despite significant progress, effective brain drug delivery remains challenging due to the lack of carriers that fully satisfy all three processes: prolonged circulation, BBB penetration, and target cell uptake. Further, while enhancing drug delivery is crucial, it's equally important to consider the safety and potential side effects of these nanomedicines, especially when utilizing viral vectors as delivery systems. Exosomes are naturally occurring nanoparticles with lipid bilayer membranes that allows to encapsulate large macromolecules as cargos. Exosomes have a natural ability to cross the BBB, partly attributed to their endogenous origin and the presence of specific surface proteins that facilitate interactions with BBB components and cellular receptors.

[0009] Despite their promising potential, challenges remain, such as: 1) Limited large-scale production: exosome production is characterized by a low yield of isolation and production. Scaling up the production process while maintaining the purity and integrity of exosomes is a complex task. 2) Lack of standardization methodology for purification and characterization. Different isolation techniques and characterization methods are employed across studies, leading to variations in the purity and quality of isolated exosomes. is no clear optimal purification technique to isolate high purity exosomes. 3) Considerable complexity and heterogeneity: the variability in size, surface proteins, and cargo content among exosomes derived from cell sources can impact their therapeutic efficacy and reproducibility. 4) Achieving precise control over drug loading within exosomes is a significant challenge.

[0010] The present invention focuses on addressing these challenges and further optimizing biomimetic-based drug delivery systems; to overcome unsatisfactory BBB targeting capacity and intracellular delivery efficiency of current nanovesicles aiming to enhance their effectiveness for therapeutic applications in clinical settings.

[0011] BRIEF DESCRIPTION OF DRAWINGS

[0012] Fig. 1 : Schematic representation of RVG-Lamp2b biomimetic nanocarriers for delivery of plasmid DNA specifically to the brain. JAWS II dendritic cells are transfected with a virus peptide (rabies viral glycoprotein (RVG) peptide) fused to a membrane glycoprotein (Lamp2b protein. The RVG-Lamp2b transfected JAWS cells serve as the source to engineer biomimetic nanocarriers by using extracted cell membrane fragments. The carriers are functionalized with a fluorescence reporter intercalated in the outer layer and a plasmid encapsulated inside.

[0013] Fig. 2 : Schematic representation of CSM synthetic protocol for cell lysis. Cell lysis is carried out through (1) osmotic shock using a hypotonic buffer and (2) repeated freezing-thawing cycles (4 cycles). Subsequently, a mechanical extrusion step is applied on the extracted membrane fragments obtained after purification by centrifugation.

[0014] Fig. 3 : JAWS derived-CSM characterization. A) Physico-chemical characterization of JAWS derived CSMs: Dynamic light scattering (DLS) measurements of the hydrodynamic diameter of CSMs dispersed in PBS buffer; Nanoparticle Tracking analysis (NTA) measurements of CSM size and concentration; Protein content determined by Bradford colorimetric assay is related to the CSM concentration determined by NTA. B) Evaluation of the RVG-Lamp2b expression on the CSM surface: Flow cytometry (FC) is utilized to characterize the presence of the specific surface markers on the CSM and the right-side-out membrane orientation of the bilayer. An anti-Lam2b antibody was used the detect the Lamp2b expression on the CSM via FC after using an APC-labeled secondary antibody. The determination of Lam2b expression on CSM derived from Lamp2b-RVG transfected JAWS cells is compared to the expression on control CSM derived from non-transfected JAWS cells (3-Fold-increased). C) The expression of Lam2b-RVG on CSM is also confirmed by SDS-PAGE Western Blot analysis.

[0015] Fig. 4 : JAWS derived-CSMs with encapsulated plasmid. A) Physico-chemical characterization of plasmid encapsulated in CSMs (CSM@plasmid): Nanoparticle Tracking analysis (NTA) measurements of CSM (with and without encapsulated plasmid) size and concentration; B) Expression of Lamp2b-RVG on CSM is confirmed by SDS-PAGE Western Blot analysis by using a primary anti-Lamp2b antibody (left panel) and a primary anti-RVG peptide antibody (right panel). In both cases, expression of Lamp2b-RVG protein is higher in transfected JAWS cells than in control JAWS cells. C) Encapsulated DNA plasmid in CSM is characterized by agarose gel electrophoresis. AMPKa1-DN, Viraquest® plasmid, control plasmid, and luciferasa-p- cDNA3 plasmid have been encapsulated in the CSMs nanocarriers. D)Fluorescence quantification of encapsulated plasmid is conducted using a DNA intercalated reporter dye (LUCS®13 green fluorescence nucleic acid stain). A loading efficiency of 94 % is achieved for plasmid encapsulation in CSM nanocarriers.

[0016] Fig. 5 : Biodistribution of RVG-Lamp2b-DC-derived CSM loaded with a luciferase-p- cDNA3. 1 : non-transfected with RVG-Lamp2b; 2 and 3: transfected with RVG-Lamp2b.

[0017] Fig. 6 : Intravenous treatment with RVG-Lamp2b-DC-derived CSM loaded with a SF1 AMPKa1-DN encoding plasmid. (A-B) Study of the body weight loss in High fat diet (HDF)-induced obese C57BL / 6 mice. (C-D) Study of the brown adipose tissue (BAT) temperature after treatment.

[0018] Fig. 7: Dynamic light scattering characterization of the nanocarrier (NC): A) mean hydrodynamic diameter for the DLS distributions in intensity (dh(l)), and number (dh(N)) of NCs before (black line) and after (red line) the extrusion steps; B) variation of the NC hydrodynamic diameter and the polydisperse index (PDI) after different number of extrusion steps.

[0019] Fig. 8: Lamp2b-RVG sEVs analyzed by NTA. Graph corresponding to Fig. 1A from Milbank E. et al. Nature Metabolism, 2021 , 3, 10, 1415-1431.

[0020] Fig. 9: Lamp2b-RVG CSMs before extrusion process analyzed by NTA.

[0021] Fig. 10: Lamp2b-RVG CSMs after the extrusion process analyzed by NTA. Three independent batches are reported.

[0022] GENERAL DEFINITIONS

[0023] It must be noted that, as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in 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.

[0024] If the term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. For instance, the term “about” means the indicated value ± 1% of its value, or the term “about” means the indicated value ± 2% of its value, or the term “about” means the indicated value ± 5% of its value, the term “about” means the indicated value ± 10% of its value, or the term “about” means the indicated value ± 20% of its value, or the term “about” means the indicated value ± 30% of its value; preferably the term “about” means exactly the indicated value (± 0%).

[0025] As used herein, the conjunctive term "and / or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and / or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or."

[0026] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention may be substituted with the term "consisting of", though less preferred.

[0027] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0028] "Nanoparticle" refers to a particle having a maximum dimension of less than 1 micron.

[0029] "Nucleic acids," "nucleic acid molecules," "oligonucleotide," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.

[0030] As used herein, the term "gene" or "coding sequence" refers to a polynucleotide sequence in vitro or in vivo that encodes a gene product. In some instances, the gene consists or consists essentially of coding sequence, that is, sequence that encodes the gene product.

[0031] As used herein, the term “AMP-activated protein kinase” (AMPK) refers to an enzyme that plays a role in cellular energy homeostasis, largely to activate glucose and fatty acid uptake and oxidation 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 a number of tissues, including the liver, brain, and skeletal muscle. In response to binding 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 modulation of insulin secretion by pancreatic betacells. AMPK is a heterotrimeric protein complex that is formed by a, p, and y subunits. As used herein, “AMPKa” refers to the subunit alpha (a) of the AMPK protein. Due to the presence of isoforms of its components, there are 12 versions of AMPK in mammals, each of which can have different tissue localizations, and different functions under different conditions. The a, p, and y subunits can also be found in different isoforms: the y subunit can exist as either the y1, y2 or y3 isoform; the p subunit can exist as either the pi or p2 isoform; and the a subunit can exist as either the a1 or a2 isoform. As used herein, “AMPKa” refers to the isoform 1 of the subunit a of AMPK protein. As used herein, the term “dominant negative AMPKal mutant protein” (AMPKa1-DN) refers to an inactive isoform of the AMPKal , preferably rat AMPKal , protein by modification of its active site with at least the point mutation D168A, wherein the amino acid position (168) is expressed with respect to the wild-type rat AMPKal sequence, preferably the wild-type rat AMPKal sequence of SEQ ID NO: 7. In the context of the present invention, “AMPKa1-DN” refers to the inactive isoform of the AMPKal , preferably rat AMPKal , protein by modification of its active site with at least the point mutations D168 with respect to the wild-type rat AMPKal sequence, preferably the wild-type rat AMPKal sequence of SEQ ID NO: 7, wherein the sequence of the AMPKa1-DN protein can be the sequence of rat AMPKal protein, or an homologous sequence of the rat AMPKal protein corresponding to the species where the treatment is applied, such as human homologous of AMPKal protein, or humanised or a codon- optimized AMPKal protein for treatment in humans. Alternatively, the rat sequence of AMPKal protein can be used to treat other species, such as humans, as it has been shown that rat AMPKa1-DN was effective in other animals such as mouse. In case of using the AMPK human sequence, the sequence may also or alternatively comprise other point mutations, such as T172A mutation.

[0032] The positions or location of the amino acid residues of a protein or a polypeptide sequence are preferably numbered sequentially starting from the first amino acid residue which would then be located at position 1. For example, a protein of 137 amino acids will have those residues numbered 1 (first amino acid residue) until 137 (the last amino acid residue). Preferably, the first position or position number 1 corresponds to the first amino acid residue located at the 5-prime (5') end of the polypeptide chain that has a nitrogen atom or a free amino group. Thus, the numbering preferably starts from the first amino acid residue at the N terminal or 5’ end of the protein or polypeptide and ends at the 3’ end or C terminal end of the protein or polypeptide. Preferably, the positions of the amino acid residues are numbered using the amino acid sequence of the translated mature protein.

[0033] As used herein, the term “nuclear receptor steroidogenic factor-1 (SF-1)” refers to a transcription factor essential for terminal differentiation and maintenance of ventromedial nucleus neuronal populations.

[0034] As used herein, the term “SF1 expressing cell or tissue” refers to cells or tissues that are able to express the SF1 transcription factor. Preferably, the SF1 expressing cell is a SF1 expressing neuron located in the VMH region.

[0035] As used herein, "pharmaceutically acceptable carrier" or “pharmaceutically acceptable diluent” means 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 pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: 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; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulphur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thio sulphate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone. The terms “sequence identity” or “percent identity” in the context of two or more nucleotide sequences, polypeptide sequences or proteins sequences refers to two or more sequences or subsequences that are the same (“identical”) or have a specified percentage of nucleotide or amino acid residues that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm, preferably BLAST alignment tool, or alternatively, by visual inspection. The “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. Calculation of percent identity includes determination of the optimal alignment between two or more sequences. Alignment can take into account insertions and deletions (i.e. “gaps”) in each of the sequences to be tested, such as, without limitation, in the non-coding regions of nucleic acids and truncations or extensions of polypeptide sequences. Computer programs and algorithms such as the Basic Local Alignment Search Tool (BLAST) may be used to determine the percent identity. BLAST is one of the many resources provided by the U.S. National Center for Biotechnology Information. Because the genetic code is degenerate, and more than one codon can encode a given amino acid, coding regions of nucleic acids are considered identical if the nucleic acids encode identical polypeptides. Thus, percent identity could also be calculated based on the polypeptide encoded by the nucleic acid. Percent identity could be calculated based on full length consensus genomic sequences or on a fraction of the genomic sequence, such as for example without limitation on individual open reading frames (ORFs).

[0036] "Percent (%) amino acid sequence identity" with respect to proteins or polypeptides described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the 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, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full-length of the sequences being compared.

[0037] Preferably, the “percentage of identity” as used herein is decided in the context of a local alignment, i.e., it is based on the alignment of regions of local similarity between nucleobase sequences, contrary to a global alignment, which aims to align two sequences across their entire span. Thus, in the context of the present invention, percentage identity is calculated preferably only based on the local alignment comparison algorithm.

[0038] By “statistically significant” or “significant” is referred herein as the determination by an analyst that the results in the data are not explainable by chance alone. Statistical hypothesis testing is the method by which the skilled person makes this determination. This test provides a p-value, which is the probability of observing results as extreme as those in the data, assuming the results are truly due to chance alone. A p-value of 0.1 or lower (preferably 0.05, 0.01, 0.001 or lower) is considered herein to be statistically significant.

[0039] DESCRIPTION OF THE EMBODIMENTS

[0040] Broadly, it is an object of the present invention to provide a biomimetic nanocarrier capable of brain targeting and BBB crossing. One of the main challenges for clinical gene therapy applications is the development of gene therapy vehicles for specifically and safely diffuse delivery to the brain. The invention provides an innovative nanoformulation utilizing a specially engineered brain-targeting biomimetic nanocarrier for delivering nucleic acids to the brain.

[0041] The methods of the invention described the fabrication of a nanocarrier based on cellular membrane fragments extracted from dendritic cells. These cells have been genetically modified to express a fusion protein consisting of a neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b) to improve the BBB targeting capabilities (Fig. 1). Lamp2b protein has adhesion and signal transduction functions while RVG peptide enables BBB crossing through its binding to the nicotinic acetylcholine receptor (nAChR).

[0042] JAWS dendritic murine cells (DCs) expressing RVG-Lamp2b are used to extract cell membrane fragments by freeze-thaw method in combination with a hypotonic buffer. The freezing process cause the formation of ice crystals, leading to the disruption of cell membranes, and subsequent thawing allows for the release of membrane fragments (Fig. 2). These membrane fragments after sonication and extrusion processes are self-assembled to form nanovesicles of 150-200 nm size in diameter (Fig. 3). These tagged biomimetic cell derived nanocarriers (called herein cellsomes, or CSM) are used for delivery of plasmid DNA specifically to the mouse brain. As a proof of concept, a plasmid DNA encoding an AMP-activated protein kinase alpha 1 (AMPKal) dominant negative mutant (AMPKa1-DN, Viraquest®) targeted by a specific promoter to hypothalamic steroidogenic factor 1 (SF1) neurons was selected as a cargo and encapsulated in the RVG-Lamp2b derived DC-CSMs nanoparticles. AMPKal has been shown to be effective in reducing obesity in treated animals. The plasmid is encapsulated in the nanoformulation by an extrusion process (Fig. 4).

[0043] As shown in Fig. 6, intravenous treatment of obese mice with RVG-Lamp2b-DC- derived CSM loaded with a SF1 AMPKa1-DN encoding plasmid showed a marked food-independent body weight loss in the experimental group (Figure 6A-B), which was associated with an increase in brown adipose tissue (BAT) temperature during the next 3 days (Figure 60) and also average BAT temperature during the same treatment period (Figure 6D). Notably uncoupling protein 1 (LICP1; the main thermogenic protein) protein levels were also augmented in the treated mice. This evidence indicates that IV treatment with VG-Lamp2b-DC-derived CSM loaded with a SF1 AMPKa1-DN ameliorates obesity in association with increased BAT function.

[0044] In view of this, biomimetic nanocarriers using cell membrane fragments that selfassemble into nanocarriers sized at 200 nm, referred to as Cellsomes (CSMs), were generated. These cells are genetically modified to express a specific targeted protein, enabling the generation of blood-brain barrier-targeted nanocarriers.

[0045] These engineered biomimetic nanocarriers offers several advantages:

[0046] Scaling-up the production: enabling the generation of substantial quantities of the nanocarriers. Increasing the number of cells in 2D cell cultures (using cell culture multi flask) allow us to increase the concentration of synthesized nanocarriers. In addition the last step of synthetic protocol (the extrusion step) have been improved with the use of Jacketed Liposome Extruder ( typically used for liposome production in compliance with FDA and GMP sanitary standards in pharmaceuticals). This scalability is crucial for meeting the demands of large-scale applications, such as those required for clinical settings.

[0047] Control size and composition: High control of size distribution and highly monodisperse nanoparticle suspension. High control ratio of protein composition integration with phospholipids. The genetic modification of cells also enables precise control over the expression of specific proteins and motifs on the final surface of the nanocarrier. Enhanced stability in Physiological Media: These nanocarriers provide better stability in physiological media for longer times resulting in an extended circulation time within the body. This increased stability is vital for ensuring the effective delivery of therapeutic payloads to the intended target areas.

[0048] Immune tolerance when using autologous-derived nanovesicles. Utilizing specific cell derived nanovesicles contributes to immune tolerance, minimizing the risk of immunogenic reactions. This property enhances the safety profile of the nanocarriers, particularly in the context of personalized medicine where the patient's own cells are employed in the production process.

[0049] Natural targeting property of producing cells in combination with the specificity motifs conferred by the expressed targeted protein, enhances the precision of drug delivery, especially to the challenging target site of the blood-brain barrier.

[0050] Delivery of macromolecules: These biomimetic nanocarriers demonstrate the capability to effectively encapsulate large macromolecules, such as plasmids. It also allows to protect them from degradation.

[0051] In view of the above, the present invention provides in a first aspect a population of cell-derived nanoparticles (also called herein the nanoparticles of the invention or cell- somes). In an embodiment, the nanoparticles (NPs) of the invention are isolated. In the context of the present invention, the term "isolated" indicates that the population of the NPs of the invention is not within the environment or cell culture where they were produced. The NPs or population thereof of the invention has been substantially separated from surrounding environment or cell culture. The term isolated also encompasses a population of NPs that have been removed from the environment or ell culture, e.g., from the supernatant or conditioning media, from which they originated.

[0052] In an embodiment, the population of NPs of the invention is substantially pure or enriched in NPs and comprises at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of NPs of the invention. In an embodiment, the population is pure in NPs of the invention.

[0053] In an embodiment, the population of NPs is a homogeneous population. By “homogeneous” is meant that each NP in the population of NPs is characterized by having substantially the same particle size or diameter, that is, for being substantially monodisperse. As used herein, “monodisperse” refers to NPs possessing a narrow average particle size distribution (PSD). By “particle size distribution” (PSD) is meant herein an index (means of expression) indicating what sizes (particle size) of particles are present in what proportions (relative particle amount as a percentage where the total amount of particles is 100%) in the sample particle group to be measured. Preferably, particle size distribution can be measured from a statistically valid analysis of thousands of particles analyzed by nanoparticle tracking analysis (NTA), and it refers to the standard deviation of the fitting.

[0054] In an embodiment, each NP of the population of NPs is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical among them in the population of NPs. By “identical” is meant that the NPs are substantially similar in their characteristics, including shape, composition, magnetic properties, coating and functionalization. Preferably, each NP of the population of NPs is at least 85%, 90%, 92%, 95%, 98%, 100% identical among them, more preferably 95% identical.

[0055] In an embodiment, each NP of the populations of NPs have a particle size distribution with a polydispersity index (PDI) of less than 0.4, 0.3, 0.25, 0.2, preferably less than 0.2, preferably as determined by dynamic light scattering. The particle size and PDI of NP can be determined using numerous commercially available instruments and methods known by the skilled artisan.

[0056] In an embodiment, the NPs of the invention are spherical or round-shaped, and have diameter of between 100-500 nm, preferably between 100-400 nm, more preferably between 150-250 nm, even more preferably between 120-160 nm, most preferably 200 nm or about 200 nm. The size and diameter of the NPs may be determined by electron microscopy or by Nanoparticle tracking analysis (NTA). The hydrodynamic diameter of the NPs may be determined by dynamic light scattering (DLS).

[0057] Further, each NP of the invention comprises at least (a) a surface and (b) at least a cargo of interest. Each of said elements will be explained in detail below:

[0058] (a) The surface of the cell-derived NPs of the invention:

[0059] In an embodiment, the (a) surface of each of the NPs of the invention comprise at least one or more fragments of cellular membranes (a.1), preferably mammalian cellular membranes. Preferably, the surface of each NP comprises plasma cell membranes and / or any intracellular membrane fragment from cell organelles. The cellular membrane may be derived from a cell component or cell organelle including, but not limited to, an exo- some, a secretory vesicle, a synaptic vesicle, an endoplasmic reticulum (ER), a Golgi apparatus, a mitochondrion, a vacuole or a nucleus.

[0060] The term "cell membrane" or “cellular membrane” as used herein refers to a biological membrane enclosing or separating structure acting as a selective barrier, within or around a cell or an emergent viral particle. The cellular membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. The cellular membrane comprises a phospholipid uni- or bilayer, and optionally associated proteins and carbohydrates. As used herein, the cellular membrane refers to a membrane obtained from a naturally occurring biological membrane of a cell or cellular organelles, or one derived therefrom.

[0061] Preferably, the NPs or population thereof of the invention are generated by fragmenting the cell membranes of a population of cells (also called herein donor cells). In an embodiment, the donor cells are mammalian cells, preferably human cells or murine cells, preferably selected from the list of erythrocytes, platelets, tumour cells, mesenchymal cells, fibroblasts, macrophages, monocytes, dendritic cells, and NKs; more preferably immune cells. Preferably, the donor cells are mammalian immune cells, more preferably antigen presenting cells, even more preferably dendritic cells. More preferably, the donor cells are immune system cell, preferably immature cells from the immune system of a mammal, more preferably immature mammalian dendritic cells.

[0062] In an embodiment, the donor cell is a mammalian cell with low immunogenicity, preferably an immature immune cell, preferably an immature antigen presenting cell, most preferably an immature dendritic cell or monocyte. By “immature” is referred herein as a cell that is not activated or biologically active, or that does not present activation markers or molecule in the surface. An immature dendritic cell has different morphological phenotype than the mature one. Immature dendritic cells have a round and smooth surface, while mature cells, such as mature dendritic cells, have a rough surface with multiple pseudopodia. Immature dendritic cells produce large quantities of exosomes devoid of T-cell activators such as MHC-II, CD80 and CD86. Thus, in an embodiment, the donor cell, preferably the immature dendritic cell, does not express activation markers, preferably T-cell activator 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 an embodiment, the donor cell is an immature antigen presenting cell characterized by having a statistically significant reduced expression of at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of at least one T-cell activator molecule in comparison to the expression of said T-cell activator molecule in a mature antigen presenting cell. The percentage of reduction of said T-cell activator molecule can be measured by techniques known in the art, e.g. by flow cytometry or RT-PCR. Thus, in an embodiment, the population NPs of the invention are positive for cellular markers, preferably cellular immature dendritic cell (iDC) markers.

[0063] The donor cell may be a primary cell or an immortalized cell. In an embodiment, the donor cell is an immature immortalized cell, preferably an immature immortalized dendritic cell or monocyte, most preferably the JAWS II cell line from the American Type Culture Collection CRL-11904 ATCC; Manassas, VA, USA or a cell generated from said JAWS II cell line. This cell product is an ATCC manufactured and accessioned progeny of ATCC CRL-11904 cited in US Pat. No. 5,648,219.

[0064] As explained above, the NPs of the invention are preferably obtained by fragmenting one or more cells, preferably mammalian cells. Thus, their surface will comprise the same composition as the cell membrane of the cells from which are obtained. This means that the surface of the NPs or population thereof of the invention may have other marker or molecules, including proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate.

[0065] The surface of the NPs of the invention further comprises (a.2) at least one fusion protein comprising or consisting of the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b). This fusion protein is called herein after RVG-Lamp2b fusion protein.

[0066] Hence, the NPs of the invention comprise or are loaded with, preferably mainly at their outer membrane, one or more RVG-Lamp2b fusion proteins. By “fusion protein” is referred herein as to a protein formed by at least two domains, wherein the at least two domains have been joined, one after the other, so that they are synthetized 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 comprised or consisting of the fusion protein are the RVG peptide and the Lamp2b protein. In an embodiment, the domains of a fusion protein may be linked by a linker peptide. “Linker peptide” as used herein is a short peptide sequence that is located between the domains of a fusion protein. Link- er peptides are placed to provide the two domains comprised in the fusion protein with movement flexibility. In the context of the present invention, the linker peptide has at least one amino acid residue, preferably at least two consecutive amino acid residues, optionally 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20 more amino acid residues. The linker peptide includes flexible linkers, rigid linkers, and in vivo cleavable linkers.

[0067] In an embodiment, the RVG-Lamp2b fusion protein comprises, consists or consists essentially of SEQ ID NO: 5, or an amino acid sequence with 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: 5. In an embodiment, the RVG-Lamp2b fusion protein is encoded by a nucleotide sequence comprising or consisting of SEQ ID NO: 6, or a nucleotide sequence with 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: 6.

[0068] The population of NPs described herein are thus characterized by comprising in their surface at least one Lamp2b-RVG fusion protein. For that purpose, a Lamp2b-RVG encoding plasmid can be transfected in the donor cells, so that the donor cells transiently express one or more Lamp2b-RVG fusion proteins, wherein, preferably, such fusion protein comprises an amino acid sequence with at least 95% sequence identity with SEQ ID NO: 5.

[0069] Preferably, the fusion protein is at a concentration range of between 5-200 pg, preferably 10 - 100 pg, fusion protein per mg of total protein. Preferably, the content of fusion protein is in the range of 1-20%, preferably 1-10%, by weight (w / w) relative to the total weight of the protein content on the NP of the invention.

[0070] Thus, the NPs of the invention are characterized by comprising:

[0071] (a) a surface comprising:

[0072] (a.1) one or more fragments of isolated cellular membranes, and (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0073] (b) at least one cargo of interest.

[0074] Preferably, the NPs of the invention are characterized by comprising:

[0075] (a) a surface comprising: (a.1) one or more fragments of isolated cellular membranes, and (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0076] (b) at least one cargo of interest, wherein the one or more fragments of isolated cellular membranes are obtained from immune mammalian cells, preferably immature mammalian dendritic cells.

[0077] Preferably, the NPs of the invention have a size of between 150 nm to 200 nm in diameter and a PDI of less than 0.2, and are characterized by comprising:

[0078] (a) a surface comprising:

[0079] (a.1) one or more fragments of isolated cellular membranes, and (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0080] (b) at least one cargo of interest, wherein the one or more fragments of isolated cellular membranes are obtained from immune mammalian cells, preferably immature mammalian dendritic cells, wherein the fusion protein comprises or consists of SEQ ID NO: 5.

[0081] (b) The cargo of interest of the cell-derived NPs of the invention:

[0082] As explained above, the NPs or population thereof of the invention comprise at least (b) a cargo of interest. Said cargo of interest may be any biomolecule of interest, such as proteins, peptides, lipids, nucleic acids, or any combination thereof. Preferably, the cargo of interest is a biomolecule or a drug.

[0083] In an embodiment, the cargo of interest is a biomolecule that is releasable. A trigger for releasing the releasable cargo from the NPs of the invention includes, but is not limited to, contact between, the NP and a target cell, tissue, organ or subject, or a change of an environmental parameter, such as the pH, ionic condition, temperature, pressure, and other physical or chemical changes, surrounding the nanoparticle. In certain embodiments, the releasable cargo comprises one or more therapeutic agent, prophylactic agent, diagnostic or marker agent, prognostic agent, e.g., an imaging marker, or any combination thereof. In certain embodiments, the releasable cargo is located within or on the inner core of each of the NPs of the invention. In other embodiments, the releasable cargo is located between the inner core and the outer surface of each of the NP of the invention. In yet other embodiments, the releasable cargo is located within or on the outer surface of the each of the NP of the invention. A trigger for releasing the releasable cargo from the inventive nanoparticle includes, but is not limited to, a contact between the NP and a target cell, tissue, organ or subject, or a change of an environmental parameter, such as the pH, ionic condition, temperature, pressure, and other physical or chemical changes, surrounding the NP.

[0084] Preferably, the cargo of interest is at least one nucleic acid, such as a RNA or DNA molecule, or any RNA / DNA hybrid. Preferably, the cargo of interest is at least one DNA molecule, more preferably a DNA plasmid. Preferably, the biomolecule, preferably nucleic acid, of interest, is mainly located in the core of the NPs of the invention.

[0085] In an embodiment, the cargo of interest is a nucleic acid, preferably a DNA nucleic acid, encoding an AMP-activated protein kinase alpha 1 (AMPKal) dominant negative mutant (also called herein “AMPKa1-DN”). In an embodiment, the AMPKa1-DN is operably linked to, or expressed under the control of, a tissue-specific promoter. Preferably, the tissue-specific promoter is the hypothalamic steroidogenic factor 1 (SF1) promoter.

[0086] A coding sequence and a promoter are said to be operably linked when they are linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. For example, the dominant negative AMPKcH mutant protein (AMPKa1-DN) is operably linked to the SF1 promoter so that the expression levels of the AMPKa1-DN are regulated by the SF1 promoter.

[0087] In an embodiment, the AMPKa1-DN mutant protein encoded by the DNA comprised in the NPs of the invention has at least the mutation or amino acid substitution D168A, wherein the amino acid numbering is expressed with respect to the AMPKal protein of rat, preferably with respect to the wild-type rat AMPKal sequence of SEQ ID NO: 7. In an embodiment, the nucleotide or amino acid sequence of the AMPKa1-DN protein is the rodent, preferably mouse, sequence. In another embodiment, the nucleotide or amino acid sequence of the AMPKa1-DN sequence is the human homologue sequence, or a humanized or human codon-optimized version of the mouse sequence. Please note that here and thorough the whole document, the amino acid residue positions are preferably numbered sequentially or consecutively starting from the N terminal end of the protein or polypeptide. It is also noted that the amino acid numbering, and therefore the number of the amino acid substitution that results in AMPKa1-DN mutant protein as defined herein, may change within different AMPKal proteins. For example, the substitution D168A in SEQ ID NO: 7 equals to substitution D169A in SEQ ID NO: 1 (AMPKa1-DN mutant protein amino acid sequence with Myc-Tag and G- linker), and substitution D156A in SEQ ID NO: 9 (AMPKa1-DN mutant protein amino acid sequence without Myc-Tag and G-linker). Thus, it is to be understood that, regardless of the numbering where said Aspartic acid is placed in the sequence of the AMPKal protein, and if it is changed for a different amino acid residue, one can arrive at the AMPKa1-DN mutant protein as defined herein. Preferably, the Aspartic acid to be mutated in the sequence of the AMPKal to generate the AMPKa1-DN mutant protein is the Aspartic acid of the conservative sequence “NAKIADFGLS" Preferably, said AMPKa1-DN mutant protein is capable of impairing or decreasing the activity of the cells' endogenous functional counterpart (AMPKal wild-type), resulting in a decrease of the pACC phosphorylation levels in a treated cell, preferably in the VMH area, but not in other SF-1 expressing tissues such as adrenal, pituitary and testis.

[0088] In an embodiment, the AMPKa1-DN mutant protein encoded by the nucleic acid, preferably DNA, comprised in the NPs of the invention comprises, consists, or consists essentially of SEQ ID NO: 7, or an amino acid sequence with 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: 7, with the proviso that the amino acid residue in position number 168 in SEQ ID NO: 7, which corresponds with an Aspartic acid, is substituted by an amino acid residue that is not Aspartic acid, preferably substituted by Alanine (mutation D168A). In an embodiment, the AMPKa1-DN mutant protein encoded by the nucleic acid, preferably DNA, comprised in the NPs of the invention comprises, consists, or consists essentially of SEQ ID NO: 7, or an amino acid sequence with 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: 7, with the proviso that the amino acid in position 168 is a Alanine (substitution D168A).

[0089] In an embodiment, the AMPKa1-DN mutant protein encoded by the nucleic acid, preferably DNA, comprised in the NPs of the invention comprises, consists, or consists essentially of SEQ ID NO: 1 , or an amino acid sequence with 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: 1 with the proviso that the amino acid in position 169 is a Alanine (substitution D to A). In an embodiment, the AMPKa1-DN mutant protein encoded by the nucleic acid, preferably DNA, comprised in the NPs of the invention comprises, consists, or consists essentially of SEQ ID NO: 9, or an amino acid sequence with 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: 9, with the proviso that the amino acid in position 156 is a Alanine (substitution D to A).

[0090] In an embodiment, the at least one polynucleotide encoding for the AMPKa1-DN mutant protein comprises, consists, or consists essentially of SEQ ID NO: 2, or a nucleotide sequence with 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: 2.

[0091] In an embodiment, the steroidogenic factor 1 (SF1) promoter sequence also comprised in the nucleic acid, preferably DNA, comprised in the NPs of the invention comprises, consists, or consists essentially of SEQ ID NO: 3, or a nucleotide sequence with 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.

[0092] In an embodiment, the nucleic acid molecule comprised in the NPs of the invention encoding an AMPKa1-DN mutant protein operably linked and under the control of the SF1 promoter comprises, consists, or consists essentially of SEQ ID NO:4, or a nucleotide sequence with 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.

[0093] Thus, designing a DNA, such as a plasmid, encoding for AMPK-DN under the control of SF-1 (SF1-AMPK-DN) will allow the AMPK-DN to be expressed only in the SF1 expressing cells located in the VMH. Once the NPs of the invention are fused with the target cells, the AMPKa1-DN mutant protein would only be expressed in the target cell. Several commercial kits are available for loading nucleic acids into NPs and the skilled person would be familiar with them.

[0094] Preferably, the cargo content is in the range of 0.1-2% by weight (w / w) relative to the total weight of the NPs. In an embodiment, the population of NPs of the invention as defined in the first aspect or any of its embodiments are obtained or obtainable following a method (also called herein the method of the present invention) comprising the steps of:

[0095] (1) providing a population of genetically engineered cells (i.e., the donor cells defined above) that express in their surface the fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0096] (2) disrupting the cells of (1) and isolating the membrane fragments of said cells, and

[0097] (3) assembling the cell membrane fragments of step (2) to provide the population of cell-derived NPs of the invention.

[0098] Each of the steps of the method of the present invention are detailed below:

[0099] Step (1) comprises or consists of providing a population of genetically engineered cells that express in their surface one or more of the fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b.

[0100] As explained above, in a preferred embodiment, the NPs are produced by fragmenting the cell membranes of one or more donor cells. Said donor cells are characterized by expressing the RVG-Lamp2b fusion protein on their surface, so that the resulting NPs comprise cell membranes comprising said RVG-Lamp2b fusion protein. As explained above, the donor cells are mammalian cells, preferably human cells or murine cells, more preferably immune cells, preferably immature dendritic cells. By “genetically engineered” or “genetically modified” is referred herein that the cells are modified to transiently or permanently express one or more of the fusion proteins as defined herein, enabling the generation of blood-brain barrier- targeted nanocarriers. Particularly, genetically modification methods include modifications at the genomic or episomal level through human-mediated intervention involving the introduction of nucleic acid se- guences, including but not limited to DNA or RNA, by means of non-natural technigues (transfection, transduction, genome editing or modification, etc).

[0101] Thus, step (1) of the method of the invention comprises preparing a culture of the donor cells, wherein said cells are modified to express the RVG-Lamp2b fusion protein on their cell membrane. Methods for culturing cells are known in the art. Methods for modifying cells to express them a specific fusion protein are also known in the art and in- elude transfection, transduction, and electroporation method. Preferably, the donor cells are transfected or transduced to deliver a DNA molecule encoding for the RVG- Lampb2 fusion protein operably linked to a promoter that is specific for said donor cell or that is a ubiquitous promoter and thus a functional promoter in said donor cells. Upon transfection or transduction, the donor cells are cultured under suitable conditions to be metabolically active and thus to transcribe and express the RVG-Lampb2 fusion protein on their surface.

[0102] As a result of step (1) of the method of the present invention, a culture of donor cells expressing RVG-Lampb2 fusion protein on their cell membranes is obtained. This resulting product is now subjected to step (2) of the method of the present invention.

[0103] In an embodiment, the cells of step (1) are harvested, washed and trypsinized before proceeding with step (2). Methods for harvesting, washing and trypsinizing cells are known in the art.

[0104] Step (2) disrupting the cells of (1) and isolating the membrane fragments of said cells

[0105] This step is a fragmentation-isolation step where the donor cells of step (1) are disrupted or lysed (sub-step 2.i) and their cell membrane fragments are subsequently purified (sub-step 2.ii).

[0106] The cell disruption / lysis (sub-step 2.i) may be carried out using mechanical, physical, chemical and / or biological methods. Examples of mechanical disruption methods include subjecting the donor cells to bead mill, dounce homogenizer, HPH, ultrasonication and / or microwaves. Examples of physical disruption methods include subjecting the donor cells to an osmotic shock. Examples of thermal disruption methods include subjecting the donor cells to freezing - thawing cycles. Examples of chemical disruption methods include subjecting the donor cells to acid hydrolysis and / or surfactant treatment. Examples of biological disruption methods include subjecting the donor cells to lytic enzymes.

[0107] In an embodiment, the cell disruption / lysis (sub-step 2. i) is carried out by lysing the cell membranes of the donor cells using a combination of physical lysing methods with mechanical or thermal lysing method. In an embodiment cell disruption / lysis (sub-step 2. i) is carried out by lysing the cell membranes of the donor cells by subjecting the donor cells to an osmotic shock combined with freeze-thaw cycles (physical combined with thermal lysing methods) or subjecting the produced cells to an osmotic shock combined with subjecting them to a dounce homogenizer (physical combined with mechanical lysing methods).

[0108] Preferably, the physical lysing methods of osmotic shock is performed by adding to the donor cells a hypotonic solution that provides the osmotic pressure required to cell swelling. The concentration of salt surrounding the cell is suddenly decreased hence the cell membrane becomes permeable to water due to the osmosis effect. As a result, transient openings are created in the cell membrane to empty the cell from its cellular components.

[0109] Preferably, the thermal methods of freeze-drying are performed by subjecting the cells to at least one, preferably 4, cycles of freezing in liquid nitrogen and thawing the solution. Repeated cycles of freezing and thawing steps lyses cells through ice crystal formation inside the cell which helps in rupturing the cell membranes. Preferably, the freezing in liquid nitrogen is performed during at least 1 minute, and the thawing is performed at 37 °C.

[0110] Preferably, the mechanical method of using a dounce homogenizer. The subjecting cells resuspended in the hypotonic lysing buffer are preferably passed at least 30 40, 50, preferably 60, times through the dounce homogeneizer (with tight-fitting pestle).

[0111] The cell fragmentation sub-step 2.i. can be carried out in the presence of protease inhibitors. The cell fragmentation sub-step 2.i. may also comprise washing substeps, and / or steps of sonicating the cells.

[0112] The resulting product of the fragmentation sub-step 2. ii is a mixture of fragmented cell membranes and other cell components (organelles, proteins, nucleic acids, etc.). This resulting product is now subjected to an isolation or purification step, also known as sub-step 2. ii.

[0113] This isolating step 2.ii comprises or consists of separating or purifying the fragmented cell membranes from the rest of the cellular components. Separation methods are known in the art and include, e.g., centrifugation methods. Thus, after sub-step 2.i, the fragmented cell membranes are isolated or purified from the rest of the cell components preferably by centrifugation. Preferably, this isolation step comprises centrifuging the resulting cell fragments of sub-step 2.i. Preferably, at least two rounds of centrifu- gation are completed. Preferably, the first round of centrifugation includes centrifuging the cell fragments at 700 g for at least 10 mins, and discarding the nucleic or whole cells. Preferably, the second round of centrifugation includes centrifugation of the supernatant obtained from the first centrifugation round at 15000 g for 30 min at 4 °C.

[0114] Thus, preferably, step (2) comprises isolating the membrane fragments of the genetically modified cells of step (1) in a process comprising the steps of:

[0115] (2.i) lysing the cell membranes using mechanical, physical, chemical and / or biological methods, and

[0116] (2.ii) purifying or isolating the cell membrane fragments, preferably by centrifugation.

[0117] Thus, preferably, step (2) comprises isolating the membrane fragments of the genetically modified cells of step (1) in a process comprising the steps of:

[0118] (2.i) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0119] (2.ii) purifying or isolating the cell membrane fragments, preferably by centrifugation.

[0120] More preferably, step (2) comprises isolating the membrane fragments of the genetically modified cells of step (1) in a process comprising the steps of:

[0121] (2.i) subjecting the cells obtained from step (1) to an osmotic shock (physical lysing method) combined with at least one, preferably four, freeze-drying cycles (thermal lysing methods), and

[0122] (2.ii) purifying or isolating the cell membrane fragments, preferably by centrifugation.

[0123] Also preferably, step (2) comprises isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0124] (2.i) subjecting the cells obtained from step (1) to an osmotic shock (physical lysing method) combined with subjecting the cells to a douncer homogenizer (mechanical lysing method), and

[0125] (2.ii) purifying or isolating the cell membrane fragments, preferably by centrifugation.

[0126] The resulting product after sub-step 2.ii is an isolated or purified mixture of cell membrane fragments, including but not limited to plasma cell membrane and intracellular membranes from cell organelles. This resulting product is now subjected to step (3) of the method of the present invention.

[0127] Step (3) comprises or consists of assembling the isolated cell membrane fragments of step (2) to provide the cell-derived population of NPs of the invention.

[0128] This step is an assembly step in which the isolated cell membranes fragments resulted after step (2) are assembled to generate a population of homogeneous cell-derived NPs of the invention.

[0129] Step (3) in turn comprises the sub-steps 3.i and 3.ii. Sub-step 3.i comprises or consists of adding to the product of step 2.ii a cargo of interest, so that it is included inside the yet-to-be formed NPs of the invention. Sub-step 3.i may be performed by mixing the resulting product of step (2) with the cargo of interest, for instance with the protein or nucleic acid of interest.

[0130] Once the mixture with the cargo of interest is obtained, sub-step 3.ii comprises or consists of extruding said mixture using a membrane that helps the NPs of the invention to form. Preferably, the membrane has a pore size of between 0.1 to 0.8 pm. Thus, substep 3.ii is an extruding step that comprises passing the isolated cell membrane fragments mixed with the cargo of interest through a membrane with a desired pore size depending on the application of the NPs. Preferably, the membrane is a polycarbonate membrane. Sub-step 3.ii can be performed more than one time. Preferably sub-step 3.ii is performed at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or preferably at least ten times. Preferably sub-step 3.ii is performed 10 times.

[0131] In this invention, it is especially preferred that a polycarbonate membrane with a pore size of 0.8 pm is used, and that the extruding step 3. ii is performed between 7-12 or 8- 12 times, preferably 10 times. This way, a homogeneous and monodisperse population of NPs are obtained, with a size of 150 - 200 nm and a polydispersity index of less than 0.2, as shown in the Examples.

[0132] In an embodiment, the population of NPs of the invention can be stored for months in a lyophilized form.

[0133] Preferred embodiments of the first aspect are: In an embodiment, the each of the homogeneous population of cell-derived NPs comprises:

[0134] (a) a surface comprising:

[0135] (a.1) one or more fragments of isolated cellular membranes, and

[0136] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0137] (b) at least one cargo of interest, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0138] (1) providing a population of immune cells, preferably dendritic cells, that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0139] (2) isolating the membrane fragments of the cells in a process comprising the steps of:

[0140] (2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0141] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0142] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0143] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0144] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least seven, preferably ten times.

[0145] In an embodiment, the homogeneous population of cell-derived NPs are characterized by having a size of between 150 nm to 200 nm in diameter, wherein each NP comprises:

[0146] (a) a surface comprising:

[0147] (a.1) one or more fragments of isolated cellular membranes, and

[0148] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0149] (b) at least one cargo of interest, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0150] (1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, (2) isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0151] (2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0152] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0153] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0154] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0155] (3.11) extruding the product of step (3.i) using a membrane with a pore size of between 0.1 to 0.8 pm, wherein step (3.ii) is performed at least seven, preferably ten, times.

[0156] In an embodiment, the homogeneous population of cell-derived NPs are characterized by having a size of between 150 nm to 200 nm in diameter and a PDI of less than 0.2, wherein each NP comprises:

[0157] (a) a surface comprising:

[0158] (a.1) one or more fragments of isolated cellular membranes, and

[0159] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0160] (b) at least one cargo of interest, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0161] (1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0162] (2) isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0163] (2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0164] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0165] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0166] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0167] (3.11) extruding the product of step (3.i) using a membrane with a pore size of between 0.1 to 0.8 pm. In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of about 200 nm in diameter, wherein each NP comprises:

[0168] (a) a surface comprising:

[0169] (a.1) one or more fragments of isolated cellular membranes, and

[0170] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0171] (b) at least one cargo of interest, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0172] (1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0173] (2) isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0174] (2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0175] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0176] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0177] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0178] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least seven, preferably ten, times.

[0179] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of about 200 nm in diameter and a PDI of less than 0.2, wherein each NP comprises:

[0180] (a) a surface comprising:

[0181] (a.1) one or more fragments of isolated cellular membranes, and

[0182] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0183] (b) at least one cargo of interest, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of: (1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0184] (2) isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0185] (2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0186] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0187] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0188] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0189] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm.

[0190] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of about 200 nm in diameter and a PDI of less than 0.2, wherein each NP comprises:

[0191] (a) a surface comprising:

[0192] (a.1) one or more fragments of isolated cellular membranes, and

[0193] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0194] (b) at least one cargo of interest, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0195] (1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0196] (2) isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0197] (2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0198] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0199] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0200] (3.1) adding to the product of step (2. ii) the cargo of interest, and (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein the cargo content is in the range of 0.1-2% by weight (w / w) relative to the total weight of the NP, and wherein the fusion protein is at a concentration range of between 10 - 100 pg fusion protein per mg of total protein.

[0201] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of between 150 nm to 200 nm in diameter and a PDI of less than 0.2, wherein each NP comprises:

[0202] (a) a surface comprising:

[0203] (a.1) one or more fragments of isolated cellular membranes, and

[0204] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0205] (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0206] (1) providing a population of immature dendritic cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0207] (2) isolating the membrane fragments of the immature dendritic cells in a process comprising the steps of:

[0208] (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0209] (2.11) purifying the cell membrane fragments by centrifugation,

[0210] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0211] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0212] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, and wherein the cargo content is in the range of 0.1-2% by weight (w / w) relative to the total weight of the NP, and wherein the fusion protein is at a concentration range of between 10 - 100 pg fusion protein per mg of total protein.

[0213] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of between 150 nm to 200 nm in diameter, wherein each NP comprises:

[0214] (a) a surface comprising: (a.1) one or more fragments of isolated cellular membranes, and

[0215] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0216] (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0217] (1) providing a population of cells that comprise or express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0218] (2) isolating the membrane fragments of the cells in a process comprising the steps of:

[0219] (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0220] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0221] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0222] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0223] (3.11) extruding the product of step (3.i) using a membrane with a pore size of between 0.1 to 0.8 pm, wherein step (3.ii) is performed at least seven, preferably ten, times.

[0224] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of between 150 nm to 200 nm in diameter, wherein each NP comprises:

[0225] (a) a surface comprising:

[0226] (a.1) one or more fragments of isolated cellular membranes, and

[0227] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0228] (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0229] (1) providing a population of dendritic cells, preferably immature dendritic cells, that comprise or express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, (2) isolating the membrane fragments of the cells in a process comprising the steps of:

[0230] (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0231] (2.11) purifying the cell membrane fragments by centrifugation,

[0232] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0233] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0234] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least seven, preferably ten, times.

[0235] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of between 150 nm to 200 nm in diameter and a PDI of less than 0.2, wherein each NP comprises:

[0236] (a) a surface comprising:

[0237] (a.1) one or more fragments of isolated cellular membranes, and

[0238] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0239] (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0240] (1) providing a population of immature dendritic cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0241] (2) isolating the membrane fragments of the immature dendritic cells in a process comprising the steps of:

[0242] (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0243] (2.11) purifying the cell membrane fragments by centrifugation,

[0244] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0245] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0246] (3.11) extruding the product of step (3. i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least ten times. In an embodiment, the each of the homogeneous population of cell-derived NPs comprises:

[0247] (a) a surface comprising:

[0248] (a.1) one or more fragments of isolated cellular membranes, and

[0249] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprising or consisting of SEQ ID NO: 5, and

[0250] (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein comprising or consisting of SEQ ID NO: 1, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0251] (1) providing a population of dendritic cells, preferably immature dendritic cells, that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0252] (2) isolating the membrane fragments of the cells in a process comprising the steps of:

[0253] (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0254] (2.11) purifying the cell membrane fragments by centrifugation,

[0255] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0256] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0257] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least seven, preferably ten times.

[0258] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of between 150 nm to 200 nm, preferably 200 nm, in diameter, wherein each NP comprises:

[0259] (a) a surface comprising:

[0260] (a.1) one or more fragments of isolated cellular membranes, and

[0261] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprising or consisting of SEQ ID NO: 5, and (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein comprising or consisting of SEQ ID NO: 1, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0262] (1) providing a population of dendritic cells, preferably immature dendritic cells, that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0263] (2) isolating the membrane fragments of the cells in a process comprising the steps of:

[0264] (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0265] (2.11) purifying the cell membrane fragments by centrifugation,

[0266] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0267] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0268] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least seven, preferably ten times.

[0269] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of between 150 nm to 200 nm in diameter and a PDI of less than 0.2, wherein each NP comprises:

[0270] (a) a surface comprising:

[0271] (a.1) one or more fragments of isolated cellular membranes, and

[0272] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprising or consisting of SEQ ID NO: 5, and

[0273] (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein comprising or consisting of SEQ ID NO: 1, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0274] (1) providing a population of immature dendritic cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0275] (2) isolating the membrane fragments of the immature dendritic cells in a process comprising the steps of: (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0276] (2.11) purifying the cell membrane fragments by centrifugation,

[0277] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0278] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0279] (3.11) extruding the product of step (3.i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least ten times.

[0280] In an embodiment, the homogeneous population of cell-derived NPs comprises NPs having a size of between 150 nm to 200 nm in diameter and a PDI of less than 0.2, wherein each NP comprises:

[0281] (a) a surface comprising:

[0282] (a.1) one or more fragments of isolated cellular membranes, and

[0283] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b comprising or consisting of SEQ ID NO: 5, and

[0284] (b) at least one cargo of interest, preferably a nucleic acid encoding for the AMPKa-DN protein comprising or consisting of SEQ ID NO: 1, wherein the cell-derived NPs are obtained or obtainable by a method comprising the steps of:

[0285] (1) providing a population of immature dendritic cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0286] (2) isolating the membrane fragments of the immature dendritic cells in a process comprising the steps of:

[0287] (2.1) lysing the cell membranes by subjecting the cells to an osmotic shock followed by at least one, preferably four, freeze-thaw cycles,

[0288] (2.11) purifying the cell membrane fragments by centrifugation,

[0289] (3) assembling the isolated cell membrane fragments resulting from step 2.ii) in a process comprising the steps of:

[0290] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0291] (3.11) extruding the product of step (3. i) using a polycarbonate membrane with a pore size of 0.8 pm, wherein step (3.ii) is performed at least ten times, and wherein the cargo content is in the range of 0.1-2% by weight (w / w) relative to the total weight of the NP, and wherein the fusion protein is at a concentration range of between

[0292] 10 - 100 pg fusion protein per mg of total protein.

[0293] Compositions of the invention.

[0294] The present invention provides in a second aspect a medicament delivery system, and / or a pharmaceutical composition comprising the population of cell-derived NPs as defined in the first aspect or any of its embodiments. Preferably, the composition of the invention comprises a medicament delivery system, and / or a pharmaceutical composition comprising the population of cell-derived NPs as defined in the first aspect or any of its embodiments, and obtained or obtainable with the method of the present invention as defined above. In certain embodiments, pharmaceutical composition of the present invention further comprises one or more additional active ingredient and / or a medically or pharmaceutically acceptable carrier or excipient, that can be administered along with or in combination with the nanoparticle of the present invention.

[0295] Cosmetic compositions comprising the population of cell-derived NPs as defined in the first aspect or any of its embodiments are also part of the second aspect of the present invention.

[0296] Uses of the cell-derived NPs of the invention.

[0297] A third aspect of the invention refers to the uses of the cell-derived NPs of the invention as defined in the first aspect or any of its embodiments, or uses of the compositions as defined in the second aspect or any of its embodiments. Said NPs or compositions can be used as delivery system to bring cargos of interest to the brain of a subject in need thereof, as they are capable of crossing the BBB. Thus, in an embodiment of the third aspect of the invention, the cell-derived NPs of the invention are used as systems for delivering an active agent, such as a therapeutic and / or imaging agent, to an animal. In many embodiments, the animal may be a warm blooded animal, such as a mammal. In certain embodiments, the animal is a human.

[0298] If the cargo of interest is a biomolecule with therapeutic uses, then the cell-derived NPs of the invention can be used as a medicament or in medicine. Thus, the present invention further provides a method for treating and / or preventing a disease or condition in a subject in need using the NPs of the invention, or the pharmaceutical composition comprising the same. A therapeutic cargo of interest may be a physiologically or pharmacologically active substance that can produce a desired biological effect in a target- ed site in an animal, such as a mammal or a human. The therapeutic agent may be any inorganic or organic compound. Examples include, without limitation, peptides, proteins, nucleic acids (including siRNA, miRNA and DNA), polymers, and small molecules.

[0299] If the cargo of interest is a biomolecule with diagnostic uses, then the cell-derived NPs of the invention can be used in diagnostic method. If the cargo of interest is a biomolecule with cosmetic uses, then the cell-derived NPs of the invention can be used in the cosmetic field. If the cargo of interest is a biomolecule with catalytic activity, then the cell-derived NPs of the invention can be used in the bioreactor / biocatalysis field.

[0300] Therefore, the third aspect of the present invention provides methods of treating a subject, as well as cosmetic methods, bioreactor / biocatalysis and diagnostic methods.

[0301] In an embodiment, the cargo of interest is a nucleic acid, preferably a DNA, encoding the AMPKa1-DN that is expressed under the control of SF1 promoter, and the cell- derived NPs of the invention are used to deliver said nucleic acid to the SF-1 expressing cells. As shown in Fig. 6, mice treated with the NPs described herein displayed a marked food-independent body weight loss in the experimental group (Figure 6A-B), which was associated with an increase in brown adipose tissue (BAT) temperature during the next 3 days (Figure 6C) and also average BAT temperature during the same treatment period (Figure 6D). This data supports the use of the AMPKa1-DN loaded NPs of the invention in the treatment and / or prevention of obesity.

[0302] In some embodiments, the NPs of the invention are produced from autologous donor cells of the subject to be treated or are obtained from cells that were isolated from the subject to be treated. In other embodiments, the NPs of the invention are produced from allogeneic cells to the subject to be treated or were obtained from cells that were isolated from a donor other than the subject to be treated. In particular embodiments, the cell is a mammalian cell, e.g., a human cell. In particular embodiments, the cell is an immature dendritic cell.

[0303] In an embodiment, the uses comprise administering the population of NPs of the invention, as defined in the first aspect or any of its embodiment, to a subject or animal. Preferably, the administration route can be systemic or local. By “systemic administration routes” is referred herein the administration of the population of the NPs of the invention into the circulatory system, directly or indirectly. Systemic administration routes comprise parenteral route such as intravascular, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, or rectal. A preferred systemic route of administration is intravascular, which is herein understood as the administration within a vessel or vessels and typically includes intravenous or intraarterial administration.

[0304] Method to produce of the cell-derived NPs of the invention.

[0305] The present invention further provides in a fourth aspect, a method for making the population of cell derived NPs of the invention. This method comprises the steps (1), (2) and (3) defined above, including all their embodiments. Preferably, the method comprises the steps of:

[0306] (1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0307] (2) isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0308] (2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0309] (2.11) purifying the cell membrane fragments, preferably by centrifugation,

[0310] (3) assembling the cell membrane fragments of step 2. ii) in a process comprising the steps of:

[0311] (3.1) adding to the product of step (2. ii) the cargo of interest, and

[0312] (3.11) extruding the product of step (3.i) using a membrane with a pore size of between 0.1 to 0.8 pm.

[0313] In an embodiment cell disruption / lysis (sub-step 2.i) is carried out by lysing the cell membranes of the donor cells using a combination of physical lysing methods with mechanical or thermal lysing method. In an embodiment cell disruption / lysis (sub-step 2.i) is carried out by lysing the cell membranes of the donor cells by subjecting the donor cells to an osmotic shock combined with freeze-thaw cycles (physical combined with thermal lysing methods) or subjecting the produced cells to an osmotic shock combined with subjecting them to a dounce homogenizer (physical combined with mechanical lysing methods).

[0314] Preferably, the physical lysing methods of osmotic shock is performed by adding to the donor cells a hypotonic solution that provides the osmotic pressure required to cell swelling. Preferably, the thermal methods of freeze-drying are performed by subjecting the cells to at least one, preferably 4, cycles of freezing in liquid nitrogen and thawing the solution. Preferably, the mechanical method of using a dounce homogenizer. The subjecting cells resuspended in the hypotonic lysing buffer are preferably passed at least 30 40, 50, preferably 60, times through the dounce homogeneizer (with tight- fitting pestle).

[0315] The isolating step (2.ii) comprises or consists of separating the fragmented cell membranes from the rest of the cellular components. Thus, after sub-step (2.i), the fragmented cell membranes are isolated from the rest of the cell components preferably by centrifugation. Sub-step (3.i) may be performed by mixing the resulting product of step (2) with the cargo of interest, for instance with the protein or nucleic acid of interest. Preferably, the extrusion step (3.ii) is performed at least seven, preferably ten times.

[0316] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0317] The following items are also encompassed in the present invention:

[0318] 1. A homogeneous population of cell-derived nanoparticles having a size of between 150 nm to 200 nm in diameter, wherein each nanoparticle comprises:

[0319] (a) a surface comprising:

[0320] (a.1 ) one or more fragments of isolated cellular membranes, and

[0321] (a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and

[0322] (b) at least one cargo of interest, wherein the cell derived nanoparticles are obtained or obtainable by a method comprising the steps of:

[0323] (1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,

[0324] (2) isolating the membrane fragments of the genetically modified cells in a process comprising the steps of:

[0325] (2.i) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and

[0326] (2.ii) purifying the cell membrane fragments, preferably by centrifugation, (3) assembling the cell membrane fragments of step 2. ii) in a process comprising the steps of:

[0327] (3.i) adding to the product of step (2. ii) the cargo of interest, and

[0328] (3.ii) extruding the product of step (3.i) using a membrane with a pore size of between 0.1 to 0.8 pm.

[0329] 2. The homogeneous population of cell-derived nanoparticles according to item 2, wherein the physical lysing methods used in step 2.i) comprise subjecting the cells to an osmotic shock using a hypotonic buffer.

[0330] 3. The homogeneous population of cell-derived nanoparticles according to any one of items 1 or 2, wherein the mechanical lysing methods used in step 2.i) comprises subjecting the cells to a dounce homogenizer.

[0331] 4. The homogeneous population of cell-derived nanoparticles according to any one of items 1 to 3, wherein the thermal lysing method used in step 2.i) comprises subjecting the cells to at least one, preferably at least four, freeze-drying cycles.

[0332] 5. The homogeneous population of cell-derived nanoparticles according to any one of items 1 to 4, wherein step 3. ii) is performed at least ten times.

[0333] 6. The homogeneous population of cell-derived nanoparticles according to any one of items 1 to 5, wherein the cell membrane fragments are:

[0334] - plasma cell membrane, and / or

[0335] - any cell membrane fragment from cell organelles.

[0336] 7. The homogeneous population of cell-derived nanoparticles according to item 6, wherein the cell membrane fragments are from a mammalian cell, preferably a human cell or murine cell, more preferably an immune cell, preferably selected from the list of erythrocytes, platelets, tumor cells, mesenchymal cells, fibroblasts, macrophages, monocytes, dendritic cells, and NKs.

[0337] 8. The homogeneous population of cell-derived nanoparticles according to any one of items 1 to 7, wherein the at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b of (a.2) comprises SEQ ID NO: 5, and wherein the fusion protein is at a concentration range of between 10 - 100 pg fusion protein per mg of total protein. The homogeneous population of cell-derived nanoparticles according to any one of items 1 to 8, wherein the nanoparticles have a polydispersity index of less than 0.2. The homogeneous population of cell-derived nanoparticles according to any one of items 1 to 9, wherein the at least one cargo of interest of (b) is a biomolecule that is encapsulated in the core of the nanoparticles, wherein the biomolecule is preferably selected from the list consisting of proteins, nucleic acids, drugs, antibodies, and any combination thereof, and wherein the cargo content is in the range of 0.1-2% by weight (w / w) relative to the total weight of the nanoparticle. The homogeneous population of cell-derived nanoparticles according to item 10, wherein the biomolecule is a nucleic acid, preferably a nucleic acid encoding an AMP-activated protein kinase alpha 1 (AMPKal) dominant negative mutant (AMPKa1-DN) that is expressed under the control of a tissue-specific promoter, preferably the tissue-specific promoter of hypothalamic steroidogenic factor 1 (SF1). Cosmetic use of the homogeneous population of cell-derived nanoparticles as defined in any of items 1 to 10. A homogeneous population of cell-derived nanoparticles as defined in any of items 1 to 10, for use in diagnostics methods. A homogeneous population of cell-derived nanoparticles as defined in any of items 1 to 11 , for use in medicine. A homogeneous population of cell-derived nanoparticles as defined in any of items 1 to 11 , for use in the treatment or prevention of obesity. SEQUENCE LISTING

[0338] SEQ ID NO 1 : AMPKa1-DN mutant protein amino acid sequence.

[0339] Note: (Underlined: Myc-Tag)

[0340] (Gray highlighted: G-Linker)

[0341] (Bold and underlined: mutations D168->A wherein the amino acid numbering or position of the mutation is with respect to rat AMPKcd protein, preferably the rat AMPKcd protein of SEQ ID NO: 7).

[0342] M EQKLISEEDLGGGEKQKH DG RVKI G H Yl LG DTLGVGTFG KVKVGKH ELTG H K- VAVKILNRQKIRSLDVVGKIRREIQNLKLFRHPHIIKLYQVISTPSDIFMVMEYVSGGEL- FDYICKNGRLDEKESRRLFQQILSGVDYCHRHMVVHRDLKPENVLLDAHMNAKI- AAFGLSNMMSDGEFLRTSCGSPNYAAPEVISGRLYAGPEVDIWSSGVILYALL- CGTLPFDDDHVPTLFKKICDGIFYTPQYLN

[0343] PSVISLLKHMLQVDPMKRATIKDIREHEWFKQDLPKYLFPEDPSYSSTMID- DEALKEVCEKFECSEEEVLSCLYNRNHQDPLAVAYHLI-

[0344] IDNRRIMNEAKDFYLATSPPDSFLDDHHLTRPHPERVPFLVAETPRARHTLD-

[0345] ELNPQKSKHQGVRKAKWHLGIRSQSRPNDIMAEVCRAIKQLDYEWKWNPYYL-

[0346] RVRRKNPVTSTFSKMSLQLYQVDSRTYLLDFRSIDDEITEAKSG- TATPQRSGSISNYRSCQRSDSDAEAQGKPSEVSLTSSV- TSLDSSPVDVAPRPGSHTIEFFEMCANLIKILAQ

[0347] SEQ ID NO 2: Polynucleotide sequence encoding for the dominant negative AMP-activated protein kinase alpha 1 (AMPKcd) mutant protein.

[0348] Note: Underlined (Myc-tag sequence)

[0349] ATGGAGCAGAAGCTTATCTCCGAGGAGGACCTCGGTGGCGGCGAGAAGCAGAA- GCACGACGGGCGGGTGAAGATCGGCCACTACATCCTGGGGGACAC-

[0350] GCTGGGCGTCGGCACCTTCGGGAAAGTGAAGGTGGGCAAGCACGAGTT- GACTGGACATAAAGTTGCTGTGAAGATACTCAACCGGCAGAAGATTCGAA- GCCTGGACGTGGTCGGGAAAATCCGCAGAGAGATCCAGAACCTGAA- GCTTTTCAGGCACCCTCATATAATCAAACTGTACCAGGTCATCAGTACAC- CGTCTGATATTTTCATGGTCATGGAATATGTCTCAGGAGGAGAGCTATTTGATTA-

[0351] TATCTGTAAAAATGGAAGGTTGGACGAAAAGGAGAGTCGACGTCTGTTCCAG- CAGATCCTTTCTGGTGTGGACTATTGTCACAGGCATATGGTGGTCCACAGAGAT- TTGAAACCTGAAAACGTCCTGCTTGATGCACACATGAATGCAAAGA-

[0352] TAGCCGCCTTCGGTCTTTCAAACATGATGTCAGATGGTGAATTTTTAAGAAC- GAGCTGTGGCTCGCCCAATTATGCTGCACCAGAAGTAATTTCAGGAA- GATTGTACGCAGGCCCTGAAGTAGACATCTGGAGCAGCGGGGTCATTCTC- TATGCTTTGCTGTGTGGAACTCTCCCTTTTGATGATGACCAC-

[0353] GTGCCAACTCTTTTTAAGAAGATATGTGACGGGATATTTTATACCCCTCAGTATTT- GAATCCCTCTGTAATAAGCCTTTTGAAGCATATGCTGCAGGTAGATCCTATGAA- GAGGGCCACAATAAAAGATATCAGGGAACATGAATGGTTTAAGCAGGAC- CTTCCAAAATATCTCTTTCCTGAAGACCCGTCTTATAGTTCAAC-

[0354] CATGATTGATGATGAAGCCTTAAAAGAAGTGTGTGAGAAGTTCGAG- TGCTCAGAGGAGGAGGTCCTCAGCTGCCTGTACAACAGAAACCACCAG- GACCCACTGGCAGTTGCCTACCACCTCATAATAGACAACAGGAGAATAATGAAC- GAAGCCAAAGATTTCTACTTGGCAACAAGCCCACCCGAT-

[0355] TCTTTCCTCGATGATCACCATTTAACTCGGCCTCACCCTGAGAGAGTAC-

[0356] CATTCTTGGTTGCCGAAACACCAAGGGCCCGACACACCCTA-

[0357] GATGAATTAAACCCACAGAAATCCAAACACCAAGGCGTACGGAAGGCAAAGTGG-

[0358] CATTTGGGGATTCGAAGTCAAAGCCGACCCAATGACATCATGGCAGAAGTGTG-

[0359] TAGAGCAATCAAGCAGTTGGACTATGAATGGAAGGTTGTAAACCCCTATTATTT-

[0360] GCGTGTGCGAAGGAAGAACCCTGTGACAAGCACATTTTCCAAAATGAGTC-

[0361] TACAGCTATACCAAGTGGATAGTAGGACTTACTTATTGGAT-

[0362] TTCCGAAGTATTGATGATGAGATTACAGAAGCCAAATCAGGGACTGC-

[0363] TACTCCACAGAGATCGGGATCCATCAGCAACTATCGATCTT-

[0364] GCCAAAGGAGCGACTCCGACGCCGAGGCTCAAGGAAA-

[0365] GCCCTCAGAAGTCTCTCTTACCTCATCCGTGACCTCCCTCGACTCCTCTCCTGTT-

[0366] GACGTAGCTCCAAGACCAGGAAGTCACACGATAGAATTTTTTGAAATGTGTG-

[0367] CAAATCTAATTAAAATTCTTGCACAGTAA

[0368] SEQ ID NO 3: Steroidogenic factor 1 (SF1) polynucleotide sequence.

[0369] AAAACAAAACAAAACAAAACAAAACAAAACAAAACAAACAAACAAACAAACAAACA

[0370] AAAACCCTTCTTTCCTACCTGGTCCTAGTACCCACATAGTCCTAC-

[0371] CTGAAGTCCCTGAAGCCACACCCTTAGCCCAGCAGTCTTGGCACAACCTCAG-

[0372] TTTCCCCAGCTACCAATGGACCATATCTGCAGCTCCCAGAGAAGCCAC-

[0373] CAAAAAGGCCACACAAACCCCACCTGATGGGTTCCAC-

[0374] CATGCCATTTCTCCACACTAGCCATTCTGACTCCTCACTCAGATCTGGGACAA-

[0375] GCTGGACCACGCAGCCCAGGCAAGGACCCAGGGAGGAAGCCATTCAAGGGGA-

[0376] GAAACTCCCAGCCTGGTAAGGGAGCAGGCCATAAATCAGGTCCCAC-

[0377] TCCCACCCAGTCGCTAACAAGCCGCTGCCTATCTGCC-

[0378] TACATGGGGTCCCTGCCTCAGGCTCCCTCATCAGCCTG-

[0379] GACAGCCAGCTGGCCAAGGTCTCTCCAGTGCCTT-

[0380] GGCCTCTGCCCCCACCCAGGGCCCCCATAAAGATAGGGA-

[0381] TATTTTTTTTTCTTTTAGAAGAGTGAAAAAAGATATAGACCCAAATGAAGA-

[0382] GAAACACCAACAAAGGAGGAGAAAGGCCTGCAGAGTCACGTGGGGGCAGAGAC-

[0383] CAATTGGGCCTCCGGTGGCCCCCCCACCCACGAGGGGAGGAGGAAAGGAC-

[0384] GATCGGACAGGGCCAGTTTCCAGTCCGCCGCTGCCCGCCCGCTGCTGGGT

[0385] SEQ ID NO 4: Polynucleotide sequence encoding an AMPKa1-DN mutant protein operably linked and under the control of the SF1 promoter.

[0386] AAAACAAAACAAAACAAAACAAAACAAAACAAAACAAACAAACAAACAAACAAACA

[0387] AAAACCCTTCTTTCCTACCTGGTCCTAGTACCCACATAGTCCTAC-

[0388] CTGAAGTCCCTGAAGCCACACCCTTAGCCCAGCAGTCTTGGCACAACCTCAG-

[0389] TTTCCCCAGCTACCAATGGACCATATCTGCAGCTCCCAGAGAAGCCAC-

[0390] CAAAAAGGCCACACAAACCCCACCTGATGGGTTCCAC-

[0391] CATGCCATTTCTCCACACTAGCCATTCTGACTCCTCACTCAGATCTGGGACAA-

[0392] GCTGGACCACGCAGCCCAGGCAAGGACCCAGGGAGGAAGCCATTCAAGGGGA-

[0393] GAAACTCCCAGCCTGGTAAGGGAGCAGGCCATAAATCAGGTCCCAC-

[0394] TCCCACCCAGTCGCTAACAAGCCGCTGCCTATCTGCC-

[0395] TACATGGGGTCCCTGCCTCAGGCTCCCTCATCAGCCTG-

[0396] GACAGCCAGCTGGCCAAGGTCTCTCCAGTGCCTT-

[0397] GGCCTCTGCCCCCACCCAGGGCCCCCATAAAGATAGGGA-

[0398] TATTTTTTTTTCTTTTAGAAGAGTGAAAAAAGATATAGACCCAAATGAAGA- GAAACACCAACAAAGGAGGAGAAAGGCCTGCAGAGTCACGTGGGGGCAGAGAC- CAATTGGGCCTCCGGTGGCCCCCCCACCCACGAGGGGAGGAGGAAAGGAC- GATCGGACAGGGCCAGTTTCCAGTCCGCCGCTGCCCGCCCGCTGCTGGGTAC- CGTTTAAACTCGAGGTCGACGGTATCGATAAGCTTGA-

[0399] TATCGAATTCGCCATGGAGCAGAAGCTTATCTCCGAGGAGGAC-

[0400] CTCGGTGGCGGCGAGAAGCAGAAGCACGACGGGCGGGTGAAGATCGGCCAC- TACATCCTGGGGGACACGCTGGGCGTCGGCACCTTCGG-

[0401] GAAAGTGAAGGTGGGCAAGCACGAGTTGACTGGACATAAAGTTGCTGTGAAGA- TACTCAACCGGCAGAAGATTCGAAGCCTGGACGTGGTCGGGAAAATCCGCAGA- GAGATCCAGAACCTGAAGCTTTTCAGGCACCCTCATATAATCAAACTGTAC- CAGGTCATCAGTACACCGTCTGATATTTTCATGGTCATGGAATATGTCTCAGGAG-

[0402] GAGAGCTATTTGATTATATCTGTAAAAATGGAAGGTTGGACGAAAAGGAGAG-

[0403] TCGACGTCTGTTCCAGCAGATCCTTTCTGGTGTGGACTATTGTCACAGGCAT- ATGGTGGTCCACAGAGATTTGAAACCTGAAAACGTCCTGCTT-

[0404] GATGCACACATGAATGCAAAGA-

[0405] TAGCCGCCTTCGGTCTTTCAAACATGATGTCAGATGGTGAATTTTTAAGAAC-

[0406] GAGCTGTGGCTCGCCCAATTATGCTGCACCAGAAGTAATTTCAGGAA-

[0407] GATTGTACGCAGGCCCTGAAGTAGACATCTGGAGCAGCGGGGTCATTCTC- TATGCTTTGCTGTGTGGAACTCTCCCTTTTGATGATGACCAC-

[0408] GTGCCAACTCTTTTTAAGAAGATATGTGACGGGATATTTTATACCCCTCAGTATTT- GAATCCCTCTGTAATAAGCCTTTTGAAGCATATGCTGCAGGTAGATCCTATGAA- GAGGGCCACAATAAAAGATATCAGGGAACATGAATGGTTTAAGCAGGAC- CTTCCAAAATATCTCTTTCCTGAAGACCCGTCTTATAGTTCAAC-

[0409] CATGATTGATGATGAAGCCTTAAAAGAAGTGTGTGAGAAGTTCGAG-

[0410] TGCTCAGAGGAGGAGGTCCTCAGCTGCCTGTACAACAGAAACCACCAG-

[0411] GACCCACTGGCAGTTGCCTACCACCTCATAATAGACAACAGGAGAATAATGAAC- GAAGCCAAAGATTTCTACTTGGCAACAAGCCCACCCGAT-

[0412] TCTTTCCTCGATGATCACCATTTAACTCGGCCTCACCCTGAGAGAGTAC- CATTCTTGGTTGCCGAAACACCAAGGGCCCGACACACCCTA-

[0413] GATGAATTAAACCCACAGAAATCCAAACACCAAGGCGTACGGAAGGCAAAGTGG-

[0414] CATTTGGGGATTCGAAGTCAAAGCCGACCCAATGACATCATGGCAGAAGTGTG-

[0415] TAGAGCAATCAAGCAGTTGGACTATGAATGGAAGGTTGTAAACCCCTATTATTT- GCGTGTGCGAAGGAAGAACCCTGTGACAAGCACATTTTCCAAAATGAGTC- TACAGCTATACCAAGTGGATAGTAGGACTTACTTATTGGAT-

[0416] TTCCGAAGTATTGATGATGAGATTACAGAAGCCAAATCAGGGACTGC-

[0417] TACTCCACAGAGATCGGGATCCATCAGCAACTATCGATCTT-

[0418] GCCAAAGGAGCGACTCCGACGCCGAGGCTCAAGGAAA-

[0419] GCCCTCAGAAGTCTCTCTTACCTCATCCGTGACCTCCCTCGACTCCTCTCCTGTT- GACGTAGCTCCAAGACCAGGAAGTCACACGATAGAATTTTTTGAAATGTGTG- CAAATCTAATTAAAATTCTTGCACAGTAA

[0420] SEQ ID NO 5: Fusion protein: Neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b).

[0421] Note: (Not underlined: Lamp2b sequence)

[0422] (Underlined: RVG sequence)

[0423] MCLSPVKGAKLILIFLFLGAVQSNALIVNLTDSKGTCLYARYTIWMPEN-

[0424] PRPGTPCDIFTNSRGKRASNGSGGAEWEM N FTITYETTNQTNK- TITIAVPDKATHDGSSCGDDRNSAKIMIQFGFAVSWAVNFT-

[0425] KEASHYSIHDIVLSYNTSDSTVFPGAVAKGVHT-

[0426] VKNPENFKVPLDVIFKCNSVLTYNLTPWQKYW-

[0427] Gl H LQAFVQNGTVSKN EQVCEEDQTPTTVAPI I HTTAPSTTTTLT-

[0428] PTSTPTPTPTPTPTVGNYSIRNGNTTCLLATMGLQLNITEEKVPFIFNINPATTNFT-

[0429] GSCQPQSAQLRLN NSQI KYLDFI FAVKN EKRFYLKEVNVYM YLANGSAF- NISNKNLSFW-

[0430] DAPLGSSYMCNKEQVLSVSRAFQINTFNLKVQPFNVTKGQYSTAQECSLDDDTILI-

[0431] PIIVGAGLSGLIIVIVIAYLIGRRKTYAGYQTL

[0432] SEQ ID NO 6: Nucleotide sequence of the fusion protein made of the Neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b (Lamp2b).

[0433] Note: (Not underlined: Lamp2b sequence)

[0434] (Underlined: RVG sequence)

[0435] GCTAGCGGTCGCCACCATGTGCCTCTCTCCGGTTAAAGGCGCAAA-

[0436] GCTCATCCTGATCTTTCTGTTCCTAGGAGCCGTTCAGTCCAATGCATTGA- TAGTTAATTTGACAGATTCAAAGGGTACTTGCCTTTATGCTCGATACACCATTT- GGATGCCCGAGAATCCGAGACCAGGGACACCTTGTGACATTTTTACCAA- TAGCAGAGGGAAGAGAGCATCCAACGGGTCCGGAGGTGCAGAATGGGA-

[0437] GATGAATTTCACAATAACATATGAAACTACAAACCAAACCAATAAAACTATAAC- CATTGCAGTACCTGACAAGGCGACACACGATGGAAGCAGTTGTGGGGATGAC- CGGAATAGTGCCAAAATAATGATACAATTTGGATTCGCTGTCTCTT- GGGCTGTGAATTTTACCAAGGAA-

[0438] GCATCTCATTATTCAATTCATGACATCGTGCTTTCCTACAACACTAGTGA-

[0439] TAGCACAGTATTTCCTGGTGCTGTAGCTAAAGGAGTTCAT-

[0440] ACTGTTAAAAATCCTGAGAATTTCAAAGTTCCATTGGATGTCATCTTTAAGTGCAA- TAGTGTTTTAACTTACAACCTGACTCCTGTCGTTCAGAAATATTGGGGTATTCAC- CTGCAAGCTTTTGTCCAAAATGGTACAGTGAGTAAAAATGAACAAGTGTGTGAA- GAAGACCAAACTCCCACCACTGTGGCACCCATCATTCACACCAC-

[0441] TGCCCCGTCGACTACAACTACACTCACTCCAACTTCAACACCCAC-

[0442] TCCAACTCCAACTCCAACTCCAACCGTTGGAAACTACAGCATTAGAAATGGCAA- TACTACCTGTCTGCTGGCTACCATGGGGCTGCAGCTGAACATCACTGAGGA- GAAGGTGCCTTTCATTTTTAACATCAACCCTGCCACAACCAACTTCACCGG- CAGCTGTCAAC-

[0443] CTCAAAGTGCTCAACTTAGGCTGAACAACAGCCAAATTAAGTATCTT-

[0444] GACTTTATCTTTGCTGTGAAAAATGAAAAACGGTTC-

[0445] TATCTGAAGGAAGTGAATGTCTACATGTATTT-

[0446] GGCTAATGGCTCAGCTTTCAACATTTCCAACAAGAACCTTAGCTTCTGG- GATGCCCCTCTGGGAAGTTCTTATATGTGCAACAAAGAG-

[0447] CAGGTGCTTTCTGTGTCTAGAGCGTTTCAGATCAACACCTTTAACCTAAAGGTG- CAACCTTTTAATGTGACAAAAGGACAGTATTCTACAGCCCAGGAGTGTTCGCTG- GATGATGACACCATTCTAATACCAATTATAGTTGGTGCTGGTCTTTCAGGCTT- GATTATCGTTATAGTGATTGCTTACCTAATTGGCAGAAGAAAGACCTATGCTGGA-

[0448] TATCAGACTCTGTAACACTAAGGATCC SEQ ID NO: 7: Wild-type 5'-AMP-activated protein kinase catalytic subunit alpha-

[0449] 1 [Rattus norvegicus]. NCBI Reference Sequence: NP_062015.2

[0450] Met Arg Arg Leu Ser Ser Trp Arg Lys Met Ala Thr Ala Glu Lys Gin 1 5 10 15

[0451] Lys His Asp Gly Arg Vai Lys He Gly His Tyr He Leu Gly Asp Thr 20 25 30

[0452] Leu Gly Vai Gly Thr Phe Gly Lys Vai Lys Vai Gly Lys His Glu Leu 35 40 45

[0453] Thr Gly His Lys Vai Ala Vai Lys He Leu Asn Arg Gin Lys He Arg 50 55 60

[0454] Ser Leu Asp Vai Vai Gly Lys He Arg Arg Glu He Gin Asn Leu Lys 65 70 75 80

[0455] Leu Phe Arg His Pro His He He Lys Leu Tyr Gin Vai He Ser Thr 85 90 95

[0456] Pro Ser Asp lie Phe Met Vai Met Glu Tyr Vai Ser Gly Gly Glu Leu 100 105 110

[0457] Phe Asp Tyr lie Cys Lys Asn Gly Arg Leu Asp Glu Lys Glu Ser Arg 115 120 125

[0458] Arg Leu Phe Gin Gin He Leu Ser Gly Vai Asp Tyr Cys His Arg His

[0459] 130 135 140 Met Vai Vai His Arg Asp Leu Lys Pro Glu Asn Vai Leu Leu Asp Ala 145 150 155 160

[0460] His Met Asn Ala Lys He Al a Asp Phe Gly Leu Ser Asn Met Met Ser 165 170 175

[0461] Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro Asn Tyr Al a Al a 180 185 190

[0462] Pro Glu Vai He Ser Gly Arg Leu Tyr Ala Gly Pro Glu Vai Asp He 195 200 205

[0463] Trp Ser Ser Gly Vai He Leu Tyr Ala Leu Leu Cys Gly Thr Leu Pro

[0464] 210 215 220

[0465] Phe Asp Asp Asp His Vai Pro Thr Leu Phe Lys Lys He Cys Asp Gly 225 230 235 240 lie Phe Tyr Thr Pro Gin Tyr Leu Asn Pro Ser Vai He Ser Leu Leu 245 250 255

[0466] Lys His Met Leu Gin Vai Asp Pro Met Lys Arg Ala Thr He Lys Asp 260 265 270 lie Arg Glu His Glu Trp Phe Lys Gin Asp Leu Pro Lys Tyr Leu Phe 275 280 285

[0467] Pro Glu Asp Pro Ser Tyr Ser Ser Thr Met He Asp Asp Glu Ala Leu

[0468] 290 295 300

[0469] Lys Glu Vai Cys Glu Lys Phe Glu Cys Ser Glu Glu Glu Vai Leu Ser 305 310 315 320

[0470] Cys Leu Tyr Asn Arg Asn His Gin Asp Pro Leu Ala Vai Ala Tyr His 325 330 335

[0471] Leu lie He Asp Asn Arg Arg He Met Asn Glu Ala Lys Asp Phe Tyr 340 345 350

[0472] Leu Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp His His Leu Thr 355 360 365

[0473] Arg Pro His Pro Glu Arg Vai Pro Phe Leu Vai Ala Glu Thr Pro Arg

[0474] 370 375 380 Ala Arg His Thr Leu Asp Glu Leu Asn Pro Gin Lys Ser Lys His Gin 385 390 395 400

[0475] Gly Vai Arg Lys Ala Lys Trp His Leu Gly He Arg Ser Gin Ser Arg 405 410 415

[0476] Pro Asn Asp He Met Ala Glu Vai Cys Arg Al a He Lys Gin Leu Asp 420 425 430

[0477] Tyr Glu Trp Lys Vai Vai Asn Pro Tyr Tyr Leu Arg Vai Arg Arg Lys 435 440 445

[0478] Asn Pro Vai Thr Ser Thr Phe Ser Lys Met Ser Leu Gin Leu Tyr Gin 450 455 460

[0479] Vai Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser lie Asp Asp Glu 465 470 475 480 lie Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gin Arg Ser Gly Ser

[0480] 485 490 495 lie Ser Asn Tyr Arg Ser Cys Gin Arg Ser Asp Ser Asp Ala Glu Ala 500 505 510

[0481] Gin Gly Lys Pro Ser Glu Vai Ser Leu Thr Ser Ser Vai Thr Ser Leu 515 520 525

[0482] Asp Ser Ser Pro Vai Asp Vai Ala Pro Arg Pro Gly Ser His Thr He 530 535 540

[0483] Glu Phe Phe Glu Met Cys Ala Asn Leu lie Lys He Leu Ala Gin 545 550 555

[0484] SEQ ID NO: 8: 5'-AMP-activated protein kinase catalytic subunit alpha-1 [Rattus norvegicus] with D168A mutation with respect to SEQ ID NO: 7.

[0485] Met Arg Arg Leu Ser Ser Trp Arg Lys Met Ala Thr Ala Glu Lys Gin 1 5 10 15 Lys His Asp Gly Arg Vai Lys He Gly His Tyr He Leu Gly Asp Thr

[0486] 20 25 30

[0487] Leu Gly Vai Gly Thr Phe Gly Lys Vai Lys Vai Gly Lys His Glu Leu 35 40 45

[0488] Thr Gly His Lys Vai Ala Vai Lys He Leu Asn Arg Gin Lys He Arg 50 55 60

[0489] Ser Leu Asp Vai Vai Gly Lys He Arg Arg Glu He Gin Asn Leu Lys 65 70 75 80 Leu Phe Arg His Pro His He He Lys Leu Tyr Gin Vai He Ser Thr 85 90 95

[0490] Pro Ser Asp He Phe Met Vai Met Glu Tyr Vai Ser Gly Gly Glu Leu

[0491] 100 105 110

[0492] Phe Asp Tyr He Cys Lys Asn Gly Arg Leu Asp Glu Lys Glu Ser Arg

[0493] 115 120 125

[0494] Arg Leu Phe Gin Gin He Leu Ser Gly Vai Asp Tyr Cys His Arg His 130 135 140

[0495] Met Vai Vai His Arg Asp Leu Lys Pro Glu Asn Vai Leu Leu Asp Ala 145 150 155 160 His Met Asn Ala Lys He Ala Ala Phe Gly Leu Ser Asn Met Met Ser

[0496] 165 170 175

[0497] Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro Asn Tyr Ala Ala 180 185 190

[0498] Pro Glu Vai He Ser Gly Arg Leu Tyr Ala Gly Pro Glu Vai Asp He

[0499] 195 200 205

[0500] Trp Ser Ser Gly Vai He Leu Tyr Ala Leu Leu Cys Gly Thr Leu Pro 210 215 220

[0501] Phe Asp Asp Asp His Vai Pro Thr Leu Phe Lys Lys He Cys Asp Gly 225 230 235 240 He Phe Tyr Thr Pro Gin Tyr Leu Asn Pro Ser Vai He Ser Leu Leu 245 250 255

[0502] Lys His Met Leu Gin Vai Asp Pro Met Lys Arg Ala Thr He Lys Asp

[0503] 260 265 270

[0504] He Arg Glu His Glu Trp Phe Lys Gin Asp Leu Pro Lys Tyr Leu Phe

[0505] 275 280 285

[0506] Pro Glu Asp Pro Ser Tyr Ser Ser Thr Met He Asp Asp Glu Ala Leu 290 295 300

[0507] Lys Glu Vai Cys Glu Lys Phe Glu Cys Ser Glu Glu Glu Vai Leu Ser 305 310 315 320 Cys Leu Tyr Asn Arg Asn His Gin Asp Pro Leu Ala Vai Ala Tyr His 325 330 335 Leu lie lie Asp Asn Arg Arg lie Met Asn Glu Ala Lys Asp Phe Tyr 340 345 350

[0508] Leu Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp His His Leu Thr 355 360 365

[0509] Arg Pro His Pro Glu Arg Vai Pro Phe Leu Vai Ala Glu Thr Pro Arg 370 375 380

[0510] Ala Arg His Thr Leu Asp Glu Leu Asn Pro Gin Lys Ser Lys His Gin 385 390 395 400

[0511] Gly Vai Arg Lys Ala Lys Trp His Leu Gly lie Arg Ser Gin Ser Arg 405 410 415 Pro Asn Asp lie Met Ala Glu Vai Cys Arg Ala lie Lys Gin Leu Asp 420 425 430

[0512] Tyr Glu Trp Lys Vai Vai Asn Pro Tyr Tyr Leu Arg Vai Arg Arg Lys 435 440 445

[0513] Asn Pro Vai Thr Ser Thr Phe Ser Lys Met Ser Leu Gin Leu Tyr Gin 450 455 460

[0514] Vai Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser lie Asp Asp Glu 465 470 475 480 lie Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gin Arg Ser Gly Ser

[0515] 485 490 495 lie Ser Asn Tyr Arg Ser Cys Gin Arg Ser Asp Ser Asp Ala Glu Ala 500 505 510

[0516] Gin Gly Lys Pro Ser Glu Vai Ser Leu Thr Ser Ser Vai Thr Ser Leu 515 520 525

[0517] Asp Ser Ser Pro Vai Asp Vai Ala Pro Arg Pro Gly Ser His Thr He 530 535 540

[0518] Glu Phe Phe Glu Met Cys Ala Asn Leu lie Lys He Leu Ala Gin 545 550 555

[0519] SEQ ID NO 9: AMPKa1-DN mutant protein amino acid sequence (without the Myc-Tag peptide and the G-linker) Bold and underlined: mutations D168->A wherein the amino acid numbering or position of the mutation is with respect to rat AMPKal protein, preferably of SEQ ID NO: 7)

[0520] Met Glu Lys Gin Lys His Asp Gly Arg Vai Lys He Gly His Tyr He 1 5 10 15

[0521] Leu Gly Asp Thr Leu Gly Vai Gly Thr Phe Gly Lys Vai Lys Vai Gly

[0522] 20 25 30

[0523] Lys His Glu Leu Thr Gly His Lys Vai Ala Vai Lys He Leu Asn Arg 35 40 45

[0524] Gin Lys He Arg Ser Leu Asp Vai Vai Gly Lys He Arg Arg Glu He

[0525] 50 55 60

[0526] Gin Asn Leu Lys Leu Phe Arg His Pro His He He Lys Leu Tyr Gin 65 70 75 80

[0527] Vai He Ser Thr Pro Ser Asp He Phe Met Vai Met Glu Tyr Vai Ser 85 90 95

[0528] Gly Gly Glu Leu Phe Asp Tyr He Cys Lys Asn Gly Arg Leu Asp Glu

[0529] 100 105 110

[0530] Lys Glu Ser Arg Arg Leu Phe Gin Gin He Leu Ser Gly Vai Asp Tyr 115 120 125

[0531] Cys His Arg His Met Vai Vai His Arg Asp Leu Lys Pro Glu Asn Vai

[0532] 130 135 140

[0533] Leu Leu Asp Ala His Met Asn Ala Lys He Ala Ala Phe Gly Leu Ser 145 150 155 160

[0534] Asn Met Met Ser Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro 165 170 175

[0535] Asn Tyr Al a Al a Pro Glu Vai He Ser Gly Arg Leu Tyr Ala Gly Pro 180 185 190

[0536] Glu Vai Asp lie Trp Ser Ser Gly Vai lie Leu Tyr Ala Leu Leu Cys 195 200 205

[0537] Gly Thr Leu Pro Phe Asp Asp Asp His Vai Pro Thr Leu Phe Lys Lys 210 215 220 lie Cys Asp Gly lie Phe Tyr Thr Pro Gin Tyr Leu Asn Pro Ser Vai

[0538] 225 230 235 240 lie Ser Leu Leu Lys His Met Leu Gin Vai Asp Pro Met Lys Arg Ala

[0539] 245 250 255

[0540] Thr lie Lys Asp lie Arg Glu His Glu Trp Phe Lys Gin Asp Leu Pro 260 265 270

[0541] Lys Tyr Leu Phe Pro Glu Asp Pro Ser Tyr Ser Ser Thr Met lie Asp 275 280 285

[0542] Asp Glu Ala Leu Lys Glu Vai Cys Glu Lys Phe Glu Cys Ser Glu Glu 290 295 300

[0543] Glu Vai Leu Ser Cys Leu Tyr Asn Arg Asn His Gin Asp Pro Leu Ala

[0544] 305 310 315 320

[0545] Vai Ala Tyr His Leu lie lie Asp Asn Arg Arg lie Met Asn Glu Ala

[0546] 325 330 335

[0547] Lys Asp Phe Tyr Leu Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp 340 345 350

[0548] His His Leu Thr Arg Pro His Pro Glu Arg Vai Pro Phe Leu Vai Ala 355 360 365

[0549] Glu Thr Pro Arg Ala Arg His Thr Leu Asp Glu Leu Asn Pro Gin Lys 370 375 380

[0550] Ser Lys His Gin Gly Vai Arg Lys Ala Lys Trp His Leu Gly lie Arg

[0551] 385 390 395 400

[0552] Ser Gin Ser Arg Pro Asn Asp lie Met Ala Glu Vai Cys Arg Ala lie

[0553] 405 410 415

[0554] Lys Gin Leu Asp Tyr Glu Trp Lys Vai Vai Asn Pro Tyr Tyr Leu Arg 420 425 430

[0555] Vai Arg Arg Lys Asn Pro Vai Thr Ser Thr Phe Ser Lys Met Ser Leu 435 440 445

[0556] Gin Leu Tyr Gin Vai Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser 450 455 460

[0557] He Asp Asp Glu He Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gin

[0558] 465 470 475 480

[0559] Arg Ser Gly Ser He Ser Asn Tyr Arg Ser Cys Gin Arg Ser Asp Ser

[0560] 485 490 495

[0561] Asp Ala Glu Ala Gin Gly Lys Pro Ser Glu Vai Ser Leu Thr Ser Ser 500 505 510

[0562] Vai Thr Ser Leu Asp Ser Ser Pro Vai Asp Vai Ala Pro Arg Pro Gly 515 520 525

[0563] Ser His Thr He Glu Phe Phe Glu Met Cys Ala Asn Leu He Lys He 530 535 540

[0564] Leu Ala Gin 545

[0565] The invention is described below by the following examples, which must be considered as merely illustrative and in no case limiting of the scope of the present invention.

[0566] EXAMPLES

[0567] EXAMPLE 1 : SYNTHESIS OF CELL-DERIVED NANOCARRIERS (CSMs)

[0568] The invention provides an innovative nanoformulation utilizing a specially engineered brain-targeting biomimetic nanocarrier for delivering nucleic acids in gene therapy. We exploit naturally occurring cell surface patterns involved for cell-cell recognition and signaling to engineered biomimetic nanocarriers. Cell membrane fragments are extracted to synthesized 200 nm sized nanocarriers (Cellsome, CSMs). Particularly, we employ a dendritic cell line (JAWS II cells, CRL-3612 ATCC®) that has been transfected with a virus peptide, namely the central nervous system-specific rabies viral glycoprotein (RVG) peptide (YTIWMPENPRPGTPCDIFTNSRGKRASNG) that enables the blood brain barrier (BBB) crossing through its binding to the nicotinic acetylcholine receptor (nAChR) fused to a membrane glycoprotein (Lamp2b protein, lysosome-associated membrane protein 2, which has adhesion and signal transduction functions. JAWS dendritic cells (DCs) expressing RVG-Lamp2b are used to extract cell membrane fragments by freeze-thaw method in combination with a hypotonic buffer. The freezing process cause the formation of ice crystals, leading to the disruption of cell membranes, and subsequent thawing allows for the release of membrane fragments. These membrane fragments after sonication and extrusion processes are self-assembled to form nanovesicles of 200 nm size in diameter. These tagged biomimetic CSMs are used for delivery of plasmid DNA specifically to the mouse brain. As cargo, a plasmid DNA encoding an AMP-activated protein kinase alpha 1 (AMPKal) dominant negative mutant (AMPKa1-DN, Viraquest®) targeted by an specific promoter to hypothalamic steroidogenic factor 1 (SF1) neurons, was selected and encapsulated in the RVG-Lamp2b derived DC-CSMs NPs (Figure 1).

[0569] 1. JAW CELLS MODIFICATION

[0570] JAWS II cells, (CRL-3612, ATCC®) were incubated in 75 cm2 or 175 cm2 cell culture flasks Alpha minimum essential medium (aMEM Complete), supplemented with ribonucleosides, deoxyribonucleosides, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng / ml murine GM-CSF, with 20 % foetal bovine serum (FBS) and Penicillinstreptomycin (50 U mL-1 - 50 pg mL-1) in a humidified chamber at 37 °C under 5 % CO2. Cells were grown in their preferred environment as they approached 70 - 80 % surface coverage. Then, the cells were seeded in 6-well plate and transfected with the Lamp2b-RVG-plasmid (the plasmid encoding for Lamp2b sequence was modified with the RVG sequence inserted between Xhol and BspEI at the N terminus of Lamp2b plasmid, to express Lamp2b-RVG protein on their membrane).

[0571] 2. SYNTHESIS OF CELL-DERIVED NANOPARTICLES

[0572] The synthesis of cell-derived NP (Cellsomes, CSM) was carried out after modification of the protocol described in the literature (D0l:10.1021 / acs.nanolett.6b02786), with a procedure to obtain monodisperse vesicles with variable size ranging from 100 to 400 nm (https: / / doi.org / 10.1002 / adbi.201900260; https: / / doi.org / 10.1016 / jjcis.2023.06.015; https: / / doi.org / 10.1186 / s12951-023-02206-5). Synthesized CSMs can be obtained from different cell lines (HeLa cells, A549 cells, HEK cells; MRC-5 cells, JAWS cells, RAW cells, THP1 cells which were purchased from ATCC®; Mesenchymal cells, RBC cells, platelets, monocytes, and NK cells).

[0573] To prepare the Lamp2b-RVG-derived-CSMs, JAWS-II cells transfected with Lamp2b- RVG-plasmid (Lamp2b-RVG-derived-cells) were harvested after trypsinization for 2 min with 2 mL of 0.25 % Trypsin-EDTA. 10 mL of DM EM supplemented with 10 % FBS was added to recover the cells and transferred to a 50 mL sterile tube. The cells were collected after centrifugation at 500 g for 5 min. The collected cells (between 10 x106to 20 x 106cells) were washed with 10 mL of precooled phosphate-buffered saline (PBS, pH 7.4) and centrifuged at 600 g for 5 min.

[0574] CSMs were produced by combination of two physical disruption methods capable of breaking down cell membranes. This physical disruption process utilizes different external forces such as heat and pressure to lyse the cell. The complete cell lysis is carried out (i) by an osmotic shock produced with a hypotonic buffer and, (ii) by thermal lysis conducted by repeated freezing-thawing cycles (see Figure 2). The hypotonic solution provides the osmotic pressure required to cell swelling. The concentration of salt surrounding the cell is suddenly decreased hence the cell membrane becomes permeable to water due to the osmosis effect. As a result, transient openings are created in the cell membrane to empty the cell from its cellular components. The cell pellet was re-suspended in 10 mL of hypotonic buffer (0.25X PBS, pH 7.4) containing 1X Protease inhibitor cocktail (PIC, Sigma-Aldrich™ #P2714-BTL) and incubated in an ice bath for 10 min. Then the cell lysis was carried out using a freeze-thaw method consisting of 4 cycles of freezing in liquid nitrogen for 1 min and thawing the solution at 37 °C for 10 min. Repeated cycles of freezing and thawing steps lyses cells through ice crystal formation inside the cell which helps in rupturing the cell membranes. Finally, the solution was placed in a bath sonicator for 5 min. To purify the cell membrane fragments, the solution was subjected to several centrifugation steps. First the solution was centrifuged at 700 g for 10 min at 4 °C to discard the nuclei or whole cells. Then the cell membrane fragments remained in the supernatant were precipitated by centrifugation at 15000 g for 30 min at 4 °C. To self-assemble the membrane fragments into NCs a mechanical extrusion process was applied. The pellet was dispersed in 1 mL of 1X PBS buffer and subject to 10 cycles of extrusion by using an Avanti® Mini extruder (Avanti Polar Lipids®) with a polycarbonate membrane. Selecting a specific pore size of the membrane (ranging from 0.1 to 0.8 pm) used during the extrusion process can modulate the size distribution of the final CSMs product. It is worth noting here that the extrusion process was found to be a critical point to obtained highly monodisperse CSMs. Increasing the number of extrusion steps would narrow the size distribution of the CSMs synthesized. A set of 10 cycles of extrusion (with a 0.8 pm polycarbonate membrane) was required to obtained highly monodisperse CSMs with and hydrodynamic diameter around 200 nm.

[0575] To increase the CSMs production, higher number of cells (> 20 x 106cells) can be used and a Jacketed Liposome extruder (Genizer®, typically used for liposome production in compliance with FDA and GMP sanitary standards in pharmaceuticals) is employed to self-assembly the membrane fragments into CSMs NP instead the Avanti® Mini extruder.

[0576] The cargo (SF1-AMPKa1-DN plasmids purchased from Viraquest®) was added to 1 mL of CSMs, incubate for 10 min at 37 °C and extruded using an Avanti® Mini extruder 10 times. The solution was then purified from the excess of non-encapsulated cargo (the free non-encapsulated plasmid) by centrifugation. Other cargos can be also encapsulated from small molecules (drugs: Doxorobucin, toxin, etc.), peptides, proteins (antibodies, etc.), complex biomolecules (CRISPR / Cas) to NPs, etc.

[0577] Fluorescently labelled CSMs were produced using fluorescent phospholipids that can be intercalated in the lipidic bilayer. In particular, 1,2-Dioleoyl-sn-glycero-3- phosphoethanolamine labelled with Atto 647N or Atto 488 (DOPE Atto 647N, Sigma- Aldrich™ #42247; DOPE Atto 488 Sigma-Aldrich™ #67335) was added to the lipid bilayer of the CSMs. 1 mL of obtained CSMs solution dispersed in 1x PBS was mixed with 2-5 pL of 1 mg mL-1 of DOPE Atto 488 or DOPE Atto 647N (dissolved in dichloromethane:methanol 8:2 or dichloromethane, respectively) and sonicated for 10 min. The resulting fluorescent CSMs were extruded 10 times using an Avanti® Mini extruder and purified by ultrafiltration or size-exclusion chromatography. Other fluorescently labelled lipids such as DiO, DiD, Dil have been used as intercalated reporter in the CSM formulations.

[0578] The CSMs can be storage at 4°C for 3-5 days or they can be subjected to lyophilisation (freeze-drying) and storage in the freezer for months. 3. CHARACTERIZATION

[0579] The cell-derived nanostructures (CSMs) produced should be fully characterized. A large variety of techniques are used for characterizing the physicochemical features of CSMs: electron microscopy (EM) for morphology determination, dynamic light scattering (DLS) for hydrodynamic size and colloidal stability, Nanotracking particle analysis (NTA) for concentration and size distribution analysis, and fluorescence spectroscopy for quantification of loading of fluorescence probes or cargos. Colorimetric assay for protein (Bradford or BCA assay) and lipid (Phospholipid Quantification Assay Kit CS0001 Sigma-Aldrich) quantification are performed. In addition, proteomic techniques (SDS-PAGE, WesternBlot, mass-spectrometry) and immunofluorescence (with Flow Cytometry) can be applied to characterize the biological properties (organization and composition of the CSM surface features).

[0580] DLS is a common and robust technique used to determine the hydrodynamic diameter of NPs and the quality and monodispersity of the size distribution indicated by the polydispersity index (PDI). DLS was used to characterize the colloidal properties of the CSMs. All the samples were analysed in PBS buffer by using a DLS Malvern Zetasizer Nano ZSP (Malvern Instrument Ktd.) equipped with a 10 mW He-Ne laser operating at a wavelength of 633 nm laser and fixed scattering angle at of 173°. All measurements were carried out at 37 °C. The mean hydrodynamic diameter (dh) for the DLS distributions in intensity (dh(l)), and number (dh(N)) are registered. After the optimization of the synthetic protocol DLS measurements revealed highly monodisperse size distribution and nearly constant hydrodynamic diameter for all the CSM samples obtained from different cell types. Similar values of the hydrodynamic diameter (ca. 200 nm) for both, intensity (dh(l)) and number distributions (dh(N)), and low values of PDI (below 0.2) were obtained. DLS was also used to study the colloidal CSM stability under different media. Nanomaterials for biological applications are expected to not sedimentate or aggregate. However, aggregation after changes in the ionic force or pH of the media can occur frequently for NP dispersion. Therefore, is crucial to analyse the CSM behaviour under several conditions. The loss of CSMs stability under some conditions of pH and / or ionic force can be reflected in the increase in the hydrodynamic diameter. Several buffers at 4 different pH conditions, from the physiological pH to the lysosomal pH (7.4, 6, 5.5, and 4.5), and two complex media (cell media DMEM and phagolysosomal simulant fluid (PSF)) were chosen for the study. Since aggregation or stability loss can be a time-dependent process, the hydrodynamic diameter of the CSMs after dispersion in the corresponding buffer was measured over time (from 0 to 48 hours) by DLS. The results shown that the CSM size and therefore their colloidal stability remain unaltered in physiological conditions (PBS buffer at pH 7.4) as well as in complex media such as completed cell culture media. potential of (F)CSMs re-dispersed in miliQ water was measured with laser doppler anemometry by Malvern Zetasizer Nano ZSP (Malvern Instrument Ktd.).

[0581] NTA was used to characterize the colloidal properties of the CSMs and determine the CSMs concentration in solution. All the samples were analyzed in 1x PBS by using a NanoSight NS300 (Malvern Instrument Ktd) equipped with a 405 nm laser. All measurements were carried out at 24 °C. CSMs were diluted 1:1000 or 1 :500 in milliQ water (200 nm filtered) to a final volume of 1 mL and loaded in the measurement chamber with a flow rate of 50 pL min-1. Flow mode measurements were obtained recording 3 videos of 60 s for each measurement. The NanoSight NS300 software was used to analyze the sample (10-100 particles / frame).

[0582] After determination of the physical properties, the composition of CSMs (lipid, membrane proteins, etc.) was examined by several techniques such as proteomics, immunolabeling and flow-cytometry. Firstly, the total protein content determined in the final CSM sample is related to the number of cells used for the CSMs extraction. A colorimetric assay, the Bradford assay (or BCA assay), was used to measure the protein concentration in the sample. Secondly, the lipid presence is demonstrated by using a specific dye for lipid staining. Cell Mask®, a dye commonly used for cell membrane staining, is applied to demonstrate the lipid presence on the CSM samples, and a phospholipid colorimetric assay was used to quantify lipidic composition on the CSM samples.

[0583] Proteins on the surface are determined by SDS-PAGE Western Blot analysis. Specific antibodies for specific surface markers (for JAWS-II cells, Ab anti-Lamp2b, and Ab anti- RVG was used). Finally, the right-side-out membrane orientation of the bilayer is analysed by using and fluorescently labelled antibody that can recognize and extracellular domain of a transmembrane protein (Ab anti-Lam2b was also used to determine the right-side orientation of the CSM formation). Flow cytometry is a celltechnique that allows characterizing also the fluorescence signal of specific surface markers at the CSM surface. We can distinguish the signal of the CSMs from the background in fluorescence, forward and the side light scattering detector. CSMs analyzed by Flow cytometry, generate a distinctive side scattering signal from the background (PBS buffer). Forward (FSC) and side scattering (SSC) signals were recorded to gather information of the CSM dispersion. Fluorescence and SSC signals were recorded to gather information of the fluorescently labelled CSMs.

[0584] 4. RESULTS

[0585] Dendritic cells (DCs), JAWS II, have been engineered to express RVG-Lamp2b as surface marker. Then cell membrane fragments are extracted and after sonication and extrusion processes are self-assembled to form nanovesicles around 200 nm size in diameter (Fig. 1). Size and concentration are determined by DLS and NTA (Fig. 1A). Then, the expression of RVG-Lamp2b in the synthesized CSMs (RVG-Lma2b CSM@JAWS) is determined by immunolabeling by flow cytometry (Fig. 1B) and western blot analysis (Fig. 1C).

[0586] Then the RVG-Lamp2b-DC-derived CSMs have been used to encapsulate several plasmids (AMPKa1-DN, Viraquest® plasmid (Fig. 4), control plasmid and luciferasa-p- cDNA3 plasmid for biodistribution analysis, Fig. 5).

[0587] 5. IN VIVO STUDIES

[0588] As preliminary in vivo studies:

[0589] 1) Biodistribution analysis of RVG-Lamp2b-DC-derived CSM with luciferase-p- cDNA3 plasmid encapsulated have been carried out and the signal of the nanoformulations have been detected in the brain of nude mice (NMRI-Foxn1nu / nu) After 6h of intravenous (IV) treatment.

[0590] 2) Intravenous treatment with RVG-Lamp2b-DC-derived CSM loaded with a SF1 AMPKa1-DN encoding plasmid to obese mice.

[0591] High fat diet (HDF)-induced obese C57BL / 6 mice were intravenously treated with RVG- Lamp2b-DC-derived CSM loaded with a SF1 AMPKa1-DN encoding plasmid. The data showed a marked food-independent body weight loss in the experimental group (Figure 6A-B), which was associated with an increase in brown adipose tissue (BAT) temperature during the next 3 days (Figure 6C) and also average BAT temperature during the same treatment period (Figure 6D). Notably uncoupling protein 1 (UCP1 ; the main thermogenic protein) protein levels were also augmented in the treated mice.

[0592] Overall, this evidence indicates that IV treatment with VG-Lamp2b-DC-derived CSM loaded with a SF1 AMPKa1-DN ameliorates obesity in association with increased BAT function. EXAMPLE 2: OPTIMIZATION OF EXTRUSION STEP

[0593] To self-assemble the membrane fragments, a mechanical extrusion process was applied. The pellet was dispersed in 1 mL of PBS buffer and subject to several cycles of extrusion by using an Avanti® Mini extruder (with a polycarbonate membrane of 0.1 to 0.8 pm pore size). It is worth noting here that the extrusion process was found to be a critical point to obtained highly monodisperse nanocarriers. Increasing the number of extrusion steps would narrow the size distribution. The results depicted in Table 1 and Fig. 7 showed that a minimum of 7 cycles of extrusion was required to improve the nanocarrier monodispersity (polydisperse index, PDI, below 0.2).

[0594] Table 1 : DLS measurements of NCs after different extrusion steps.[1]Mean hydrodynamic diameter derived from the DLS distributions: dn,i and dn,N refer to the mean average hydrodynamic diameter from the intensity and number DLS distributions. PDI re- fers to polydispersity index. Standard deviation values were calculated from five measurements. EXAMPLE 3: Comparison with sEVs described in Milbank E. et al. Nature Metabolism, 2021, 3, 10, 1415-1431 and obtained with the protocol described in said paper, that is different from the method of the present invention

[0595] Cellsomes (CSMs) are produced through a combination of two physical disruption methods capable of breaking down cell membranes. This disruption process utilizes, for example, external forces such as heat and pressure to lyse the cells. Complete cell lysis may be achieved through an osmotic shock using a hypotonic buffer, followed by thermal lysis via repeated freeze-thaw cycles. After lysis, the solution undergoes an isolation step comprising, for example, several centrifugation steps to recover the cell membrane fragments.

[0596] To assemble the membrane fragments into CSMs, a mechanical extrusion process is applied. In this case, the sample underwent 10 extrusion cycles, using an Avanti® Mini Extruder (Avanti Polar Lipids®) equipped with polycarbonate membranes with 0.8 pm in pore size. It is worth noting that the extrusion step comprising at least 7 cycles is critical for obtaining highly monodisperse CSMs, as shown in example 2. Here, a set of 10 extrusion cycles was found to be the most optimal for producing CSMs with controlled hydrodynamic diameter and uniform size distribution.

[0597] NTA was used to characterize the colloidal properties of the CSMs and determine the CSMs concentration in solution. All the samples were analyzed using a NanoSight NS300 (Malvern Instrument Ktd) equipped with a 405 nm laser. All measurements were carried out at 24 °C. CSMs were diluted 1:500 in milliQ water (200 nm filtered) to a final volume of 1 mL and loaded in the measurement chamber with a flow rate of 50 pL min-1. Flow mode measurements were obtained recording 3 videos of 60 s for each measurement. The NanoSight NS300 software was used to analyze the sample (10- 100 particles / frame).

[0598] Mode SD D10 D50 D90

[0599] SAMPLE

[0600] (nm) (nm) (nm) (nm) (nm)

[0601] Exosomes (sEVs from Milbank E. et al. Nature

[0602] 142,7 93 124 167.7 290,4

[0603] Metabolism, 2021, 3, 10, 1415-1431.)

[0604] CSM before extrusion 142,6 80,4 125,3 180,5 312,8 CSM after 10 Extrusion cycles

[0605] 135.9 45,5 90,6 124,7 196,6

[0606] (Batch 1)

[0607] CSM after 10 Extrusion cycles

[0608] 136,4 43,2 94,4 128,9 184,1

[0609] (Batch 2)

[0610] CSM after 10 Extrusion cycles

[0611] 147.9 46,4 106,4 144,3 219,3

[0612] (Batch 3)

[0613] Table 2. The distribution shows: mode (the most frequent particle size detected), SD (standard deviation, reflects the size distribution spread), D10 (10% of the particles are smaller than this value), D50 (50% of the particles are smaller than this value. This represents the median of the population), D90 (90% of the particles are smaller than this value, indicating the upper boundary of the main size range).

[0614] Figure 10 shows the size distribution profiles of Cellsomes (CSMs) obtained after the lysis and extrusion process, analyzed across three independent batches. All batches display a sharp, monodisperse peak centered around 120-160 nm, indicating that the preparation method is highly reproducible. The narrow distribution and consistent peak positions confirm that the extrusion step is effective in generating uniformly sized vesicles. Notably, the minimal presence of larger particles across all batches suggests good colloidal stability and a lack of significant aggregation.

[0615] In comparison to the Lamp2b-RVG sEVs shown in Figure 8 (Graph obtained from Milbank E. et al. Nature Metabolism, 2021, 3, 10, 1415-1431) which exhibit a broader size distribution with a standard deviation (SD) above 90 nm, a D90 of 290.4 nm, and a mode of 142.7 nm, the CSMs in Figures 10 appear more uniform after the extrusion step (results before extrusion step are shown in Figure 9), with the SD reduced by approximately 50% (to around 45 nm). Overall, these results underscore the critical role of extrusion in achieving monodisperse CSM populations and demonstrate the robustness and reproducibility of the preparation protocol across multiple independent batches.

Claims

CLAIMS1. A homogeneous population of cell-derived nanoparticles having a size of between 150 nm to 200 nm in diameter, wherein each nanoparticle comprises:(a) a surface comprising:(a.1) one or more fragments of isolated cellular membranes, and(a.2) at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b, and(b) at least one cargo of interest, wherein the cell derived nanoparticles are obtained or obtainable by a method comprising the steps of:(1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,(2) isolating the membrane fragments of the genetically engineered cells in a process comprising the steps of:(2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and(2.11) purifying the cell membrane fragments, preferably by centrifugation,(3) assembling the cell membrane fragments of step 2. ii) in a process comprising the steps of:(3.1) adding to the product of step (2. ii) the cargo of interest, and(3.11) extruding the product of step (3.i) using a membrane with a pore size of between 0.1 to 0.8 pm, wherein step (3.ii) is performed at least seven times.

2. The homogeneous population of cell-derived nanoparticles according to claim 1 , wherein the physical lysing methods used in step 2.i) comprise subjecting the cells to an osmotic shock using a hypotonic buffer.

3. The homogeneous population of cell-derived nanoparticles according to any one of claims 1 or 2, wherein the mechanical lysing methods used in step 2.i) comprises subjecting the cells to a dounce homogenizer.

4. The homogeneous population of cell-derived nanoparticles according to any one of claims 1 to 3, wherein the thermal lysing method used in step 2.i) com-prises subjecting the cells to at least one, preferably at least four, freeze-drying cycles.

5. The homogeneous population of cell-derived nanoparticles according to any one of claims 1 to 4, wherein step 3. ii) is performed ten times.

6. The homogeneous population of cell-derived nanoparticles according to any one of claims 1 to 5, wherein the cell membrane fragments are:- plasma cell membrane, and / or- any cell membrane fragment from cell organelles.

7. The homogeneous population of cell-derived nanoparticles according to claim 6, wherein the cell membrane fragments are from a mammalian cell, preferably a human cell or murine cell, more preferably an immune cell, preferably selected from the list of erythrocytes, platelets, tumor cells, mesenchymal cells, fibroblasts, macrophages, monocytes, dendritic cells, and NKs.

8. The homogeneous population of cell-derived nanoparticles according to any one of claims 1 to 7, wherein the at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b of (a.2) comprises SEQ ID NO: 5, and wherein the fusion protein is at a concentration range of between 10 - 100 pg fusion protein per mg of total protein.

9. The homogeneous population of cell-derived nanoparticles according to any one of claims 1 to 8, wherein the nanoparticles have a polydispersity index (PDI) of less than 0.2.

10. The homogeneous population of cell-derived nanoparticles according to any one of claims 1 to 9, wherein the at least one cargo of interest of (b) is a biomolecule that is encapsulated in the core of the nanoparticles, wherein the biomolecule is preferably selected from the list consisting of proteins, nucleic acids, drugs, antibodies, and any combination thereof, and wherein the cargo content is in the range of 0.1-2% by weight (w / w) relative to the total weight of the nanoparticle.

11. The homogeneous population of cell-derived nanoparticles according to claim 10, wherein the biomolecule is a nucleic acid, preferably a nucleic acid encoding an AMP-activated protein kinase alpha 1 (AMPKal) dominant negative mutant (AMPKa1-DN) that is expressed under the control of a tissue-specific promoter, preferably the tissue-specific promoter of hypothalamic steroidogenic factor 1 (SF1).

12. Cosmetic use of the homogeneous population of cell-derived nanoparticles as defined in any of claims 1 to 10.

13. A homogeneous population of cell-derived nanoparticles as defined in any of claims 1 to 10, for use in diagnostics methods.

14. A homogeneous population of cell-derived nanoparticles as defined in any of claims 1 to 11, for use in medicine.

15. A homogeneous population of cell-derived nanoparticles as defined in claim 11 , for use in the treatment or prevention of obesity.

16. A method for producing an homogeneous population of cell-derived nanoparticles having a size of between 150 nm to 200 nm in diameter, the method comprising the steps of:(1) providing a population of genetically engineered cells that express in their surface at least one fusion protein comprising the neurotrophic rabies virus (RVG) peptide fused to lysosome-associated membrane protein 2b,(2) isolating the membrane fragments of the genetically engineered cells in a process comprising the steps of:(2.1) lysing the cell membranes using physical lysing methods combined with mechanical or thermal lysing methods, and(2.11) purifying the cell membrane fragments, preferably by centrifugation,(3) assembling the cell membrane fragments of step 2.ii) in a process comprising the steps of:(3.1) adding to the product of step (2. ii) the cargo of interest, and(3.11) extruding the product of step (3.i) using a membrane with a pore size of between 0.1 to 0.8 pm, wherein step (3.ii) is performed at least seven times.

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