WO2016044947A1 - Exosomes utiles pour traiter une maladie de stockage lysosomal - Google Patents

Exosomes utiles pour traiter une maladie de stockage lysosomal Download PDF

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WO2016044947A1
WO2016044947A1 PCT/CA2015/050959 CA2015050959W WO2016044947A1 WO 2016044947 A1 WO2016044947 A1 WO 2016044947A1 CA 2015050959 W CA2015050959 W CA 2015050959W WO 2016044947 A1 WO2016044947 A1 WO 2016044947A1
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protein
exosomes
lysosomal
gaa
seq
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Mark TARNOPOLSKY
Adeel SAFDAR
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Exerkine Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/04—Peptides being immobilised on, or in, an organic carrier entrapped within the carrier, e.g. gel, hollow fibre
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs

Definitions

  • the present invention generally relates to exosomes, and more particularly, to the use of exosomes to treat a lysosomal storage disease.
  • Lysosomes are cellular organelles that function as a cellular waste disposal mechanism to remove unused, damaged, or excessive macro-molecules.
  • mitochondria mitochondria
  • the lysosomal pathway is also involved in a variety of cellular processes including cell secretion, signalling cascades, energy metabolism, plasma membrane repair, and mitochondrial homeostasis.
  • these cellular processes include autophagy/mitophagy, endocytosis and phagocytosis.
  • the enzymes within lysosomes are encoded by nuclear DNA, transcribed on ribosomes in the rough endoplasmic reticulum, and subsequently targeted to the Golgi apparatus where they are packaged and released in small vesicles that ultimately fuse with endosomes to form lysosomes.
  • a mannose-6-phosphate moiety is added to the lysosomal protein in the Golgi apparatus and this is important for lysosomal targeting through an interaction with the mannose-6-phosphate receptors inside the Golgi.
  • the Golgi apparatus releases the vesicles, they fuse with late endosomes, following which the mannose-6-phosphate moieties are cleaved and the mature enzyme remains enveloped in the established lysosome.
  • Extracellular proteins can enter the cell through receptor- and non- receptor-mediated endocytosis and form early endosomes.
  • the early endosomes can then fuse together to form multi-vesicular bodies (MVBs) to become lysosomes.
  • MVBs multi-vesicular bodies
  • lysosomes are usually characterized by the presence of a membrane protein such as lysosomal-associated membrane protein (LAMP), e.g.
  • LAMP lysosomal-associated membrane protein
  • LSDs Lysosomal storage diseases
  • enzyme non-functional lysosomal protein
  • the specific genetic mutations negatively affect the ability of the hydrolytic enzymes within the lysosome to perform their allotted function, thus leading to the accumulation of the precursor products for that enzyme.
  • This aggregation of the intra-lysosomal precursor protein perpetuates downstream cellular consequences including the displacement of normal cellular contents and/or disruption of the lysosomes that can cause the release of hydrolytic enzymes into the cytosol which may damage other macromolecules crucial for cellular metabolism, redox homeostasis, and survival.
  • a lysosomal storage disease is Pompe disease that results from a genetic mutation in a protein called acid alpha-glucosidase (GAA). Mutations in the GAA protein lead to the progressive build-up of glycogen in skeletal and cardiac muscle that culminates in: (A) a severe infantile form with cardiomyopathy, respiratory failure and weakness, or (B) a late onset form which leads to muscle weakness and eventually respiratory failure.
  • GAA acid alpha-glucosidase
  • ERT operates on the premise that the pathology specific defective enzyme is replaced through direct infusion of a nascent lysosomal enzyme into circulation.
  • This infused enzyme produced via recombinant protein technology, is then taken up by mannose-6- phosphate receptors on the cell surface. Once inside the cell, the protein can be internalized through mannose-6-phosphate receptors on lysosomes, and thereby replace the defective enzyme.
  • mannose-6-phosphate receptors Once inside the cell, the protein can be internalized through mannose-6-phosphate receptors on lysosomes, and thereby replace the defective enzyme.
  • the infused enzyme is exposed to a non-native pH environment that causes it to denature, leading to a rapid elimination of the infused protein from the circulation. Therefore, the bioavailability of the infused enzyme is quite low as the total exposure to the affected tissues is limited. It has been estimated that only a small fraction (1-3%) of the infused recombinant GAA protein in Pompe disease is actually retained within skeletal muscle 2 weeks after the infusion. Secondly, there is a vast difference between different tissues with respect to their ability to respond to ERT. Skeletal muscle (especially type II fibers) is much more resistant to ERT therapy in comparison to cardiac muscle, when ERT is used in Pompe disease treatment.
  • enzyme uptake in ERT can be enhanced using a fusion protein containing GAA- insulin like growth factor 2 through a glycosylation-independent lysosomal targeting strategy (GILT-tagged, J Biol Chem. 2013 Jan 18;288(3): 1428-38). Enzyme uptake may also be enhanced using a carbohydrate-remodelled approach in which additional carbohydrate moieties are attached to the GAA to enhance the affinity of the GAA protein for the mannose-6-phosphate receptor (Zhu et al., Biochem J 389, 619-628, 2005).
  • ERT approaches One major issue with any of these ERT approaches is that present ERT can lead to the production of neutralizing (IgG) and/or allergenic (IgE) antibodies.
  • a carrier system is needed that can reduce immunogenicity of the infused ERT.
  • ERT enzymes cannot cross the blood-brain barrier.
  • This makes conventional ERT unusable for lysosomal storage diseases that affect the central nervous system e.g. Niemann-Pick C, neuronal ceroid lipofuscinosis, Tay-Sachs disease, Krabbe disease, etc.
  • Other routes of delivery for central nervous system diseases i.e. intra-cerebral or intra-ventricular delivery, pose a host of secondary adverse side effects and therapy may not be as effective.
  • exosomes may be effectively used as a vehicle to deliver a protein and/or nucleic acid to a mammal to treat pathological conditions resulting from a protein deficiency such as a lysosomal storage disease.
  • exosomes that are genetically modified to incorporate a functional lysosomal protein and/or nucleic acid encoding a functional lysosomal protein.
  • a method of increasing the amount of a lysosomal protein in lysosomes in a mammal comprising administering to the mammal a composition comprising exosomes that are genetically modified to incorporate a functional lysosomal protein and/or nucleic acid encoding a functional lysosomal protein.
  • a method of increasing the activity of a target protein in a mammal comprising administering to the mammal exosomes which are genetically modified to incorporate a functional protein and/or nucleic acid encoding a functional protein.
  • a method of treating a pathological condition in a mammal resulting from the deficiency of a protein comprising administering to the mammal exosomes genetically engineered to incorporate the protein or nucleic acid encoding the protein.
  • a method of treating a lysosomal storage disease in a mammal comprising administering to the mammal exosomes genetically engineered to incorporate a protein useful to treat the lysosomal storage disease or nucleic acid encoding the protein.
  • nucleic acid comprising a nucleotide sequence that encodes the functional lysosomal protein or precursor thereof
  • exosome according to any of paragraphs Al - A5, that comprises a nucleic acid comprising a nucleotide sequence encoding a functional lysosomal protein or precursor thereof, wherein the nucleic acid is present in a lumen of the exosome.
  • nucleic acid comprises mRNA or modified mRNA (modRNA, e.g. 5 methyl cytosine, or N6 methyladenine) encoding for a protein set forth in Table 1.
  • modified mRNA e.g. 5 methyl cytosine, or N6 methyladenine
  • exosome according to any one of paragraphs B2 - B6, further comprising at least one fusion product comprising a lysosome targeting sequence linked to an exosomal membrane marker.
  • exosome according to any one of paragraphs Al - A5 or Bl, that comprises at least one fusion product comprising a lysosome targeting sequence linked to an exosomal membrane marker.
  • exosome according to paragraph B7 or B8, wherein the exosomal membrane marker is selected from the group consisting of CD9, CD37, CD53, CD63, CD81, CD82, CD151, an integrin, ICAM-1, CDD31, an annexin, TSG101, ALIX, lysosome-associated membrane protein 1, lysosome-associated membrane protein 2, lysosomal integral membrane protein and a fragment of any exosomal membrane marker that comprises at least one intact transmembrane domain.
  • the exosomal membrane marker is selected from the group consisting of CD9, CD37, CD53, CD63, CD81, CD82, CD151, an integrin, ICAM-1, CDD31, an annexin, TSG101, ALIX, lysosome-associated membrane protein 1, lysosome-associated membrane protein 2, lysosomal integral membrane protein and a fragment of any exosomal membrane marker that comprises at least one intact transmembrane domain.
  • B10 The exosome according to any one of paragraphs B7 - B9, wherein the lysosomal targeting sequence is selected from the group consisting of lysosome-associated membrane protein 1, lysosome-associated membrane protein 2, lysosomal integral membrane protein, and a C-terminal sequence thereof comprising the sequence, G-Y-X-X-X H , where X H is one of glycine, valine, leucine, isoleucine, methionine, alanine, proline, tryptophan or phenylalanine, and X may be any amino acid. [0033] Bl l .
  • AI - A5 AI - A5
  • a pharmaceutically acceptable carrier a pharmaceutically acceptable carrier
  • composition according to paragraph CI wherein the composition is substantially free of vesicles having a diameter less than 20 nm.
  • C3. The composition according to paragraph CI or C2, wherein the composition is substantially free of vesicles having a diameter greater than 120mm.
  • composition according ton claim C4 which exhibits a zeta potential of up to 200 mV, or up to 175 mV, or up to 150 mV, or up to 140 mV, or up to 130 mV, or up to 120 mV, or up to 110 mV, or up to 100 mV.
  • Dl A method of increasing the amount of a lysosomal protein in lysosomes in a mammal, comprising administering to the mammal an exosome according to any one of paragraphs Al - B 15, or a composition according to any one of paragraphs CI - C5.
  • a method of treating a lysosomal storage disease in a mammal comprising administering to the mammal an exosome according to any one of paragraphs A1 - B 15, or a composition according to any one of paragraphs CI - C5.
  • D5. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human.
  • D6 The method or use according to paragraph D5, wherein the human has a lysosomal storage disease selected from the group consisting of Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl Ester Storage Disease, Cystinosis, Danon Disease, Fabry Disease, Farber Disease, Fucosidosis, Galactosialidosis, Gaucher Disease Type I, Gaucher Disease Type II, Gaucher Disease Type III, GMl Gangliosidosis Type I, GMl Gangliosidosis Type II, GMl Gangliosidosis Type III, GM2 - Sandhoff disease, GM2 - Tay-Sachs disease, GM2 - Gangliosidosis, AB variant, Mucolipidosis II, Krabbe Disease, Lysosomal acid lipase deficiency, Metachromatic Leukodystrophy, MPS I - Hurler Syndrome, MPS I - Scheie Syndrome, MPS I Hurler-Scheie Syndrome
  • Figure 1 graphically illustrates that GAA mRNA loaded unmodified exosomes or GAA protein-loaded modified exosomes rescue GAA deficiency in primary fibroblasts isolated from GAA knock-out mice at higher efficiency than conventional naked GAA ERT.
  • B Mean ⁇ SD of experiments in (A) independently repeated 3 times. *P ⁇ 0.05. Data were analyzed using an unpaired /-test.
  • FIG. 2 graphically illustrates that GAA mRNA loaded unmodified exosomes or GAA protein-loaded modified exosomes rescue GAA deficiency in primary myotubes isolated from GAA knock-out mice at higher efficiency than conventional naked GAA ERT.
  • B Mean ⁇ SD of experiments in (A) independently repeated 3 times. *P ⁇ 0.05. Data were analyzed using an unpaired /-test.
  • Figure 3 graphically illustrates that GAA mRNA loaded unmodified exosomes or GAA protein-loaded modified exosomes rescue GAA deficiency in primary fibroblasts isolated from Pompe patients at higher efficiency than conventional naked GAA ERT.
  • A Primary dermal fibroblasts isolated from three Pompe patients and three age/gender- matched controls, and were treated with naked GAA protein, empty exosomes (exosome control), GAA protein-loaded exosomes, or GAA mRNA-loaded exosomes for 48 hours. Cells were harvested after 48 hours and GAA activity was measured.
  • B Mean ⁇ SD of experiments in (A) independently repeated 3 times. *P ⁇ 0.05. Data were analyzed using an unpaired /-test.
  • Figure 4 graphically illustrates that GAA mRNA loaded unmodified exosomes or GAA protein-loaded modified exosomes reduced glycogen build-up in primary fibroblasts isolated from Pompe patients at higher efficiency than conventional naked GAA ERT.
  • FIG. 5 graphically illustrates that GAA protein-loaded exosomes therapy reduces body weight in GAA KO mice.
  • Figure 6 graphically illustrates that GAA protein-loaded exosome therapy increases strength and motor control vs. conventional naked GAA ERT in GAA KO mice.
  • FIG. 7 graphically illustrates that GAA protein-loaded exosome therapy increases EDL (fast-twitch muscle) and soleus (slow-twitch muscle) mass vs. conventional naked GAA ERT in GAA KO mice.
  • EDL fast-twitch muscle; white bar
  • soleus blunt- twitch muscle; red bar
  • Figure 8 graphically illustrates that GAA protein-loaded exosome therapy increases mixed fiber-type mass vs. conventional naked GAA ERT in GAA KO mice.
  • FIG. 9 graphically illustrates that GAA protein-loaded exosome therapy rescues pathogenic cardiac hypertrophy and brain mass vs. conventional naked GAA ERT in GAA KO mice.
  • A Heart and
  • Data were analyzed using an unpaired /-test.
  • FIG. 10 graphically illustrates that GAA protein-loaded exosome therapy increases tissue GAA activity vs. conventional naked GAA ERT in GAA KO mice.
  • B GAA activity in aforementioned tissues in GAA KO mice using approach in (A) vs. GAA protein-loaded exosomes ERT for 7 weeks (once a week intravenously, corresponding to 40 mg/kg GAA). *P ⁇ 0.05. Data were analyzed using an unpaired /-test.
  • FIG 11 graphically illustrates that GAA protein-loaded exosome therapy reduces tissue total glycogen content vs. conventional naked GAA ERT in GAA KO mice.
  • FIG. 12 graphically illustrates that GAA mRNA-loaded exosome therapy increases skeletal muscle, diaphragm, and heart GAA activity in GAA KO mice.
  • Littermate wildtype (WT) mice were used as controls for both treatments. *P ⁇ 0.05. Data were analyzed using an unpaired /-test.
  • Figure 14 graphically illustrates that GAA mRNA-loaded exosome therapy normalizes skeletal muscle and brain GAA gene expression in GAA KO mice.
  • FIG. 15 graphically illustrates that GAA mRNA-loaded exosome therapy reduces pathological glycogen content in heart and skeletal muscle of GAA KO mice.
  • Littermate wildtype (WT) mice were used as controls for both treatments. *P ⁇ 0.05. Data were analyzed using an unpaired t-test.
  • FIG. 16 graphically illustrates that GAA mRNA-loaded exosome therapy increases grip endurance in GAA KO mice.
  • Littermate wildtype (WT) mice were used as controls for both treatments. *P ⁇ 0.05. Data were analyzed using an unpaired t-test.
  • Figure 17 graphically illustrates that NPCl mRNA loaded exosome therapy rescues substrate accumulation in primary fibroblasts isolated from Niemann-Pick Disease Type C patient.
  • Cells were harvested after 48 hours and total cholesterol and glycosphingolipid content was measured using filipin staining. Mean ⁇ SD of experiments independently repeated 3 times. *P ⁇ 0.05. Data were analyzed using an unpaired t-test.
  • Exosomes are provided which have been genetically engineered to incorporate a lysosomal protein, and/or nucleic acid encoding a lysosomal protein.
  • exosome refers to cell-derived vesicles having a diameter of between about 40 and 120 nm, preferably a diameter of about 50-100 nm, for example, a diameter of about 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. Exosomes may be isolated from any suitable biological sample from a mammal, including but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g.
  • immature dendritic cells wild-type or immortalized
  • induced and non-induced pluripotent stem cells fibroblasts, platelets, immune cells, reticulocytes, tumour cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like.
  • cultured cell samples will be in the cell-appropriate culture media (using exosome-free serum).
  • Exosomes include specific surface markers not present in other vesicles, including surface markers such as tetraspanins, e.g.
  • Exosomes may also be obtained from a non-mammalian biological sample, including cultured non-mammalian cells.
  • exosomes from non-mammalian sources include surface markers which are isoforms of mammalian surface markers, such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles.
  • mammalian surface markers such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles.
  • the term "mammal” is meant to encompass, without limitation, humans, domestic animals such as dogs, cats, horses, cattle, swine, sheep, goats and the like, as well as non-domesticated animals such as, but not limited to, mice, rats and rabbits.
  • non-mammal is meant to encompass, for example, exosomes from microorganisms such as bacteria, flies, worms, plants, fruit/vegetables (e.g. corn, pomegranate) and yeast.
  • Exosomes may be obtained from the appropriate biological sample using a combination of isolation techniques, for example, centrifugation, filtration and ultracentrifugation methodologies.
  • the isolation protocol includes the steps of: i) exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation; ii) subjecting the supernatant from step i) to centrifugation to remove microvesicles therefrom; iii) microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) subjecting the microfiltered supernatant from step iii) to at least one round of ultracentrifugation to obtain an exosome pellet; and v) re-suspending the exosome pellet from step iv) in a physiological solution and conducting a second ultracentrifugation in a density gradient and remove the exosome pellet fraction therefrom.
  • the process of isolating exosomes from a biological sample includes a first step of removing undesired large cellular debris from the sample, i.e. cells, cell components, apoptotic bodies and the like greater than about 7-10 microns in size.
  • This step is generally conducted by centrifugation, for example, at 1000-4000x g for 10 to 60 minutes at 4 °C, preferably at 1500-2500x g, e.g. 2000x g, for a selected period of time such as 10-30 minutes, 12-28 minutes, 14-24 minutes, 15-20 minutes or 16, 17, 18 or 19 minutes.
  • a suitable commercially available laboratory centrifuge e.g.
  • Thermo- ScientificTM or Cole-ParmerTM is employed to conduct this isolation step.
  • the resulting supernatant is subjected to a second optional centrifugation step to further remove cellular debris and apoptotic bodies, such as debris that is at least about 7-10 microns in size, by repeating this first step of the process, i.e. centrifugation at 1000-4000x g for 10 to 60 minutes at 4 °C, preferably at 1500-2500x g, e.g. 2000x g, for the selected period of time.
  • the supernatant resulting from the first centrifugation step(s) is separated from the debris-containing pellet (by decanting or pipetting it off) and may then be subjected to an optional additional (second) centrifugation step, including spinning at 12,000-15, OOOx g for 30-90 minutes at 4 °C to remove intermediate-sized debris, e.g. debris that is greater than 6 microns size.
  • this centrifugation step is conducted at 14,000x g for 1 hour at 4 °C.
  • the resulting supernatant is again separated from the debris-containing pellet.
  • the resulting supernatant is collected and subjected to a third centrifugation step, including spinning at between 40,000-60,000x g for 30-90 minutes at 4 °C to further remove impurities such as medium to small-sized microvesicles greater than 0.3 microns in size e.g. in the range of about 0.3-6 microns.
  • the centrifugation step is conducted at 50,000x g for 1 hour.
  • the resulting supernatant is separated from the pellet for further processing.
  • the supernatant is then filtered to remove debris, such as bacteria and larger microvesicles, having a size of about 0.22 microns or greater, e.g. using microfiltration.
  • the filtration may be conducted by one or more passes through filters of the same size, for example, a 0.22 micron filter.
  • filters of the same or of decreasing sizes e.g. one or more passes through a 40-50 micron filter, one or more passes through a 20-30 micron filter, one or more passes through a 10-20 micron filter, one or more passes through a 0.22-10 micron filter, etc.
  • Suitable filters for use in this step include the use of 0.45 and 0.22 micron filters.
  • the microfiltered supernatant may then be combined with a suitable physiological solution, preferably sterile, for example, an aqueous solution, a saline solution or a carbohydrate-containing solution in a 1 : 1 ratio, e.g. 10 mL of supernatant to lOmL of physiological solution, to prevent clumping of exosomes during the subsequent ultracentrifugation and to maintain the integrity of the exosomes.
  • a suitable physiological solution preferably sterile, for example, an aqueous solution, a saline solution or a carbohydrate-containing solution in a 1 : 1 ratio, e.g. 10 mL of supernatant to lOmL of physiological solution, to prevent clumping of exosomes during the subsequent ultracentrifugation and to maintain the integrity of the exosomes.
  • the exosomal solution is then subjected to ultracentrifugation to pellet exosomes and any remaining contaminating microves
  • This ultracentrifugation step is conducted at 110,000- 170,000x g for 1-3 hours at 4 °C, for example, 170,000x g for 3 hours.
  • This ultracentrifugation step may optionally be repeated, e.g. 2 or more times, in order to enhance results.
  • Any commercially available ultracentrifuge e.g. Thermo-ScientificTM or BeckmanTM, may be employed to conduct this step.
  • the exosome-containing pellet is removed from the supernatant using established techniques and re-suspended in a suitable physiological solution.
  • the re-suspended exosome-containing pellet is subjected to density gradient separation to separate contaminating microvesicles from exosomes based on their density.
  • density gradients may be used, including, for example, a sucrose gradient, a colloidal silica density gradient, an iodixanol gradient, or any other density gradient sufficient to separate exosomes from contaminating microvesicles (e.g. a density gradient that functions similar to the 1.100-1.200 g/ml sucrose fraction of a sucrose gradient).
  • density gradients include the use of a 0.25-2.5 M continuous sucrose density gradient separation, e.g.
  • sucrose cushion centrifugation comprising 20-50% sucrose; a colloidal silica density gradient, e.g. PercollTM gradient separation (colloidal silica particles of 15-30 nm diameter, e.g. 30%/70% w/w in water (free of RNase and DNase), which have been coated with polyvinylpyrrolidone (PVP)); and an iodixanol gradient, e.g. 6-18% iodixanol.
  • the resuspended exosome solution is added to the selected gradient and subjected to ultracentrifugation at a speed between 110,000-170,000x g for 1-3 hours.
  • the resulting exosome pellet is removed and re- suspended in physiological solution.
  • the re-suspended exosome pellet resulting from the density gradient separation may be ready for use.
  • the density gradient used is a sucrose gradient
  • the exosome pellet is removed from the appropriate sucrose gradient fraction, and is ready for use, or may preferably be subjected to an ultracentrifugation wash step at a speed of 110,000- 170,000x g for 1-3 hours at 4 °C.
  • the density gradient used is, for example, a colloidal silica or a iodixanol density gradient, then the resuspended exosome pellet may be subjected to additional wash steps, e.g.
  • the exosome pellet from any of the centrifugation or ultracentrifugation steps may be washed between centrifugation steps using an appropriate physiological solution, e.g. saline.
  • the final pellet is removed from the supernatant and may be re-suspended in a physiologically acceptable solution for use.
  • the exosome pellet may be stored for later use, for example, in cold storage at 4°C, in frozen form or in lyophilized form, prepared using well-established protocols.
  • the exosome pellet may be stored in any physiological acceptable carrier, optionally including cryogenic stability and/or vitrification agents (e.g. DMSO, glycerol, trehalose, polyhydroxylated alcohols (e.g. methoxylated glycerol, propylene glycol), M22 and the like).
  • cryogenic stability and/or vitrification agents e.g. DMSO, glycerol, trehalose, polyhydroxylated alcohols (e.g. methoxylated glycerol, propylene glycol), M22 and the like.
  • the described exosome isolation protocol advantageously provides a means to obtain mammalian exosomes which are at least about 90% pure, and preferably at least about 95% or greater pure, i.e.
  • Exosomes isolated according to the methods described herein exhibit a high degree of stability, evidenced by the zeta potential of a mixture/solution of such exosomes, for example, a zeta potential of at least a magnitude of 30 mV, e.g.
  • zeta potential refers to the electrokinetic potential of a colloidal dispersion, and the magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles (exosomes) in a dispersion.
  • a zeta potential of magnitude 30 mV or greater indicates moderate stability, i.e. the solution or dispersion will resist aggregation, while a zeta potential of magnitude 40-60 mV indicates good stability, and a magnitude of greater than 60 mV indicates excellent stability.
  • exosomes in an amount of about 100-2000 ⁇ g total protein can be obtained from 1-4 mL of mammalian serum or plasma, or from 15-20 mL of cell culture spent media (from at least about 2 x 10 6 cells).
  • solutions comprising exosomes at a concentration of at least about 5 ⁇ g/ ⁇ L, and preferably at least about 10-25 ⁇ g/ ⁇ L may readily be prepared due to the high exosome yields obtained by the present method.
  • the term "about” as used herein with respect to any given value refers to a deviation from that value of up to 10%, either up to 10% greater, or up to 10%) less.
  • Exosomes isolated in accordance with the methods herein described which beneficially retain integrity, and exhibit a high degree of purity (being "essentially free” from entities having a diameter less than 20 nm and greater than 120 nm), stability and biological activity both in vitro and in vivo, have not previously been achieved.
  • the present exosomes are uniquely useful, for example, diagnostically and/or therapeutically, e.g. for the in vivo delivery of protein and/or nucleic acid. They have also been determined to be non- allergenic/non-immunogenic, and thus, safe for autologous, allogenic, and xenogenic use.
  • Isolated exosomes may then be genetically engineered to incorporate an exogenous protein, e.g. an exogenous protein such as a lysosomal protein, or exogenous nucleic acid encoding a selected protein, or both.
  • exogenous is used herein to refer to protein or nucleic acid originating from a source external to the exosomes.
  • Nucleic acid encoding the protein may be produced using known synthetic techniques, incorporated into a suitable expression vector using well established methods to form a protein-encoding expression vector which is introduced into isolated exosomes using known techniques, e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like.
  • the selected protein may be produced using recombinant techniques, or may be otherwise obtained, and then may be introduced directly into isolated exosomes by electroporation or transfection. More particularly, electroporation applying voltages in the range of about 20-1000 V/cm may be used to introduce nucleic acid or protein into exosomes.
  • Transfection using cationic lipid-based transfection reagents such as, but not limited to, Lipofectamine® MessengerMAXTM Transfection Reagent, Lipofectamine® RNAiMAX Transfection Reagent, Lipofectamine® 3000 Transfection Reagent, or Lipofectamine® LTX Reagent with PLUSTM Reagent, may also be used.
  • the amount of transfection reagent used may vary with the reagent, the sample and the cargo to be introduced.
  • an amount in the range of about 0.15 uL to 10 uL may be used to load 100 ng to 2500 ng mRNA or protein into exosomes.
  • Other methods may also be used to load protein into exosomes including, for example, the use of cell-penetrating peptides.
  • Exosomes isolated in accordance with the methods herein described which beneficially retain integrity, and exhibit a high degree of purity and stability, readily permit loading of exogenous protein and/or nucleic acid in an amount of at least about 1 ng nucleic acid (e.g. mRNA) per 10 ug of exosomal protein or 30 ug protein per 10 ug of exosomal protein.
  • nucleic acid e.g. mRNA
  • a protein-encoding expression vector as above described may be introduced directly into exosome-producing cells, e.g. autologous, allogenic, or xenogenic cells, such as immature dendritic cells (wild-type or immortalized), induced and non- induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumour cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like, by electroporation or transfection as described above. Following a sufficient period of time, e.g. 3-7 days to achieve stable expression of the protein, exosomes incorporating the expressed protein may be isolated from the exosome-producing cells as described herein.
  • exosomes incorporating the expressed protein may be isolated from the exosome-producing cells as described herein.
  • exosomes prior to incorporation into exosomes of a selected protein, or nucleic acid encoding the protein, exosomes may be modified to express or incorporate a target- specific fusion product.
  • the target-specific fusion product comprises a lysosome targeting sequence, linked to an exosomal membrane marker.
  • the exosomal membrane marker of the fusion product will localize the fusion product within the membrane of the exosome to enable the targeting sequence to direct the exosome to the intended target.
  • exosome membrane markers include, but are not limited to: tetraspanins such as CD9, CD37, CD53, CD63, CD81, CD82 and CD151; targeting or adhesion markers such as integrins, ICAM-1 and CDD31; membrane fusion markers such as annexins, TSG101, ALIX; and other exosome transmembrane proteins such as LAMP (lysosome-associated membrane protein), e.g. LAMP 1 or 2, and LFMP (lysosomal integral membrane protein). All or a fragment of an exosome membrane marker may be utilized in the fusion product provided that any fragment includes a sufficient portion of the membrane marker to enable it to localize within the exosome membrane, i.e. the fragment comprises at least one intact transmembrane domain to permit localization of the portion of the membrane marker into the exosomal membrane.
  • tetraspanins such as CD9, CD37, CD53, CD63, CD81, CD82 and CD151
  • the target-specific fusion product also includes a cell or organelle targeting sequence such as a lysosome targeting sequence, i.e. a protein or peptide sequence which facilitates the targeted lysosomal uptake of the exosome.
  • a lysosome targeting sequence i.e. a protein or peptide sequence which facilitates the targeted lysosomal uptake of the exosome.
  • suitable lysosomal targeting sequences include, but are not limited to, the lysosomal targeting sequence of LAMP and LFMP e.g. the C-terminal sequence thereof, comprising the sequence, G-Y-X-X-X H , where
  • X H is a hydrophobic residue such as glycine, valine, leucine, isoleucine, methionine, alanine, proline, tryptophan or phenylalanine, and X may be any amino acid.
  • examples of lysosomal targeting sequences derived from the C-terminal sequence of LAMP or LIMP include sequences such as GYQSV (SEQ ID NO: 1), GYQTL (SEQ ID NO: 2), GYQTI (SEQ ID NO: 3), GYEVM (SEQ ID NO: 4), GYEQF (SEQ ID NO: 5), AYQAL (SEQ ID NO: 6), NYTHL (SEQ ID NO: 7), GYQRI (SEQ ID NO: 8), GYDQL (SEQ ID NO: 9), GYKEI (SEQ ID NO: 10), and GYRHV (SEQ ID NO: 11).
  • GYQSV SEQ ID NO: 1
  • GYQTL SEQ ID NO: 2
  • GYQTI SEQ ID NO: 3
  • GYEVM SEQ ID NO: 4
  • GYEQF SEQ ID NO: 5
  • AYQAL SEQ ID NO: 6
  • NYTHL SEQ ID NO: 7
  • GYQRI
  • the lysosomal targeting sequence may be a dileucine-based motif, e.g. DXXLL (SEQ ID NO: 12), or [DE]XXXL[LI] (SEQ ID NO: 13), such as SFHDD SDEDLL (SEQ ID NO: 14), EE SEERDDHLL (SEQ ID NO: 15), GYHDD SDEDLL (SEQ ID NO: 16), ASVSLLDDELM (SEQ ID NO: 17), AS SGLDDLDLL (SEQ ID NO: 18), VQNP S ADRNLL (SEQ ID NO: 19), NALSWLDEELL (SEQ ID NO:20), TERERLL (SEQ ID NO: 21), SETERLL (SEQ ID NO: 22), TDRTPLL (SEQ ID NO: 23), and EETQPLL (SEQ ID NO: 24).
  • DXXLL SEQ ID NO: 12
  • [DE]XXXL[LI] SEQ ID NO: 13
  • SFHDD SDEDLL SEQ ID NO:
  • lysosomal targeting sequences include; ITGFSDDVPMV (SEQ ID NO: 25), DERAPLI (SEQ ID NO: 26), NEQLPML (SEQ ID NO: 27) and DDQRDLI (SEQ ID NO: 28).
  • the targeting sequence may be linked to the exosomal membrane marker with a hydrophobic linker comprising 4-5 hydrophobic amino acid moieties, including one or more of glycine, valine, leucine, isoleucine, methionine, alanine, proline, tryptophan or phenylalanine, which are the same or different.
  • the hydrophobic linker may include 4-5 glycine residues, or 4-5 of any one of the other hydrophobic amino acids, or may include a mixture of 2 or more hydrophobic amino acids.
  • Exosomes incorporating the lysosome-specific fusion product may be produced using recombinant technology.
  • an expression vector encoding the target-specific fusion product is introduced by electroporation or transfection into exosome-producing cells isolated from an appropriate biological sample.
  • the desired lysosomal protein, nucleic acid encoding the protein or both may be introduced into isolated exosomes incorporating a lysosome-targeting fusion product (modified lysosome-targeting exosomes) as previously described, using electroporation or transfection methods. Addition to the exosome of both the desired lysosomal protein and nucleic acid encoding the same lysosomal protein may increase delivery efficiency of the protein.
  • Exosomes genetically engineered to incorporate a protein, and/or nucleic acid encoding the protein may be used to deliver the protein and/or nucleic acid to mammal in vivo in the treatment of a pathological condition or disease in which the protein is defective or absent to upregulate the activity of the protein and thereby treat the disease.
  • exosomes incorporating a lysosomal protein including exosomes modified to include a lysosome-targeting fusion product and unmodified exosomes (i.e.
  • exosomes not including a lysosome-targeting fusion product may be used to deliver a protein (or nucleic acid encoding the protein) to lysosomes in vivo in the treatment of a pathological condition or disease such as a lysosomal storage disease (LSD), a disease in which a protein is defective or missing in lysosomes.
  • LSD lysosomal storage disease
  • Examples of lysosomal storage diseases that may be treated using the present engineered exosomes are set out in Table 1 below, and identify the protein required to treat the disease and the NCBI (National Centre for Biotechnology Information) reference which provides mRNA transcript sequence information, as well as protein sequence information.
  • GM2 - Gangliosidosis AB variant GM2 activator (GM2A) NM 000405.4
  • Mucolipidosis I Sialidosis alpha-N -acetyl neuraminidase NM 001304450.1
  • Salla Disease sialin (sialic acid transporter) NM 012434.4
  • the lysosomal protein for incorporation into exosomes according to the invention may be a functional native mammalian lysosomal protein, including for example, a protein from human and non-human mammals, or a functionally equivalent protein.
  • the term "functionally equivalent” is used herein to refer to a protein which exhibits the same or similar function (retains at least about 30% of the activity of native lysosomal protein) to the native protein, and includes all isoforms, variants, recombinant produced forms, and naturally-occurring or artificially modified forms, i.e. including modifications that do not adversely affect activity and which may increase cell uptake, stability, activity and/or therapeutic efficacy.
  • the term “functionally equivalent” also refers to nucleic acid, e.g. mRNA, DNA or cDNA, encoding a lysosomal protein, and is meant to include any nucleic acid sequence which encodes a functional lysosomal protein, including all transcript variants, variants that encode protein isoforms, variants due to degeneracy of the genetic code, artificially modified variants, and the like. Protein modifications may include, but are not limited to, one or more amino acid substitutions (for example, with a similarly charged amino acid, e.g.
  • substitution of one amino acid with another each having non-polar side chains such as valine, leucine, alanine, isoleucine, glycine, methionine, phenylalanine, tryptophan, proline
  • substitution of one amino acid with another each having basic side chains such as histidine, lysine, arginine
  • substitution of one amino acid with another each having acidic side chains such as aspartic acid and glutamic acid
  • substitution of one amino acid with another each having polar side chains such as cysteine, serine, threonine, tyrosine, asparagine, glutamine), additions or deletions
  • modifications to amino acid side chains addition of a protecting group at the N- or C- terminal ends of the protein, addition of oligosaccharides such as phosphorylated mannopyranosyl oligosaccharides including at least one mannose-6-phosphate (e.g.
  • Mannose-6-phospate M6P
  • phosphopentamannose bi- and tri- antennary mannopyranosyl oligosaccharides
  • bis-M6P and tri-M6P mannopyranosyl oligosaccharides
  • galactose mannose
  • N-acetylglucosamine and fucose
  • fusion products e.g. with Fc peptide
  • Suitable modifications will generally maintain at least about 70% sequence similarity with the active site and other conserved domains of native lysosomal protein, and preferably at least about 80%, 90%, 95% or greater sequence similarity.
  • Nucleic acid modifications may include one or more base substitutions or alterations, addition of 5' or 3' protecting groups, and the like, preferably maintaining significant sequence similarity, e.g. at least about 70%, and preferably, 80%, 90%, 95% or greater.
  • Modified or unmodified exosomes including a selected protein, such as a lysosomal protein, or nucleic acid encoding the protein in accordance with the invention may be formulated for therapeutic use by combination with a pharmaceutically or physiologically acceptable carrier.
  • a pharmaceutically or physiologically acceptable carrier means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable for physiological use.
  • the selected carrier will vary with intended utility of the exosome formulation.
  • exosomes are formulated for administration by infusion or injection, e.g.
  • a medical-grade, physiologically acceptable carrier such as an aqueous solution in sterile and pyrogen-free form, optionally, buffered or made isotonic.
  • the carrier may be distilled water (DNase- and RNase-free), a sterile carbohydrate-containing solution (e.g. sucrose or dextrose) or a sterile saline solution comprising sodium chloride and optionally buffered.
  • Suitable sterile saline solutions may include varying concentrations of sodium chloride, for example, normal saline (0.9%), half-normal saline (0.45%), quarter-normal saline (0.22%), and solutions comprising greater amounts of sodium chloride (e.g. 3%-7%, or greater).
  • Saline solutions may optionally include additional components, e.g. carbohydrates such as dextrose and the like. Examples of saline solutions including additional components, include Ringer's solution, e.g.
  • PBS phosphate buffered saline
  • TRIS hydroxymethyl) aminomethane hydroxymethyl) aminomethane
  • EBSS Hank's balanced salt solution
  • SSC standard saline citrate
  • HBS HEPES-buffered saline
  • GBSS Gey's balanced salt solution
  • the present exosomes are formulated for administration by routes including, but not limited to, oral, intranasal, enteral, topical, sublingual, intra-arterial, intramedullary, intrauterine, intrathecal, inhalation, ocular, transdermal, vaginal or rectal routes, and will include appropriate carriers in each case.
  • exosomes may be formulated in normal saline, complexed with food, in a capsule or in a liquid formulation with an emulsifying agent (honey, egg yolk, soy lecithin, and the like).
  • Exosome compositions for topical application may be prepared including appropriate carriers.
  • Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents, anti-oxidants and other preservatives may be added to the composition to prevent microbial growth and/or degradation over prolonged storage periods.
  • Modified and unmodified exosomes according to the present invention are useful in a method to treat a pathological condition involving a defective/missing protein, or a condition involving lack of expression of a protein, e.g. a lysosomal storage disease.
  • the terms "treat”, “treating” or “treatment” are used herein to refer to methods that favorably alter a pathological condition such as a lysosomal storage disease or other disease in which there is a protein deficiency, including those that moderate, reverse, reduce the severity of, or protect against, the progression of the target disease.
  • a therapeutically effective amount of modified or unmodified exosomes for example, carrying a selected lysosomal protein, or nucleic acid encoding a selected lysosomal protein, are administered to a mammal.
  • therapeutically effective amount is an amount of exosome required to treat the condition, while not exceeding an amount which may cause significant adverse effects. Exosome dosages that are therapeutically effective will vary on many factors including the nature of the condition to be treated as well as the particular individual being treated.
  • Appropriate exosome dosages for use include dosages sufficient to result in an increase in activity of the target protein in the patient by at least about 10%, and preferably an increase in activity of the target protein of greater than 10%, for example, at least 20%), 30%), 40%), 50%o or greater.
  • the dosage may be a dosage of exosome that delivers from about 0.1 mg/kg to about 100 mg/kg, such as 0.1-50 mg/kg, or 0.1-10 mg/kg, of the desired protein, or an exosome dosage that delivers a sufficient amount of nucleic acid to yield about 0.1 mg/kg to about 100 mg/kg, such as 0.1-50 mg/kg, or 0.1-10 mg/kg, of the desired protein.
  • the dosage of mRNA encoding a particular protein may be in the range of about 1 ug/kg to 1 mg/kg to treat a given disease such as an LSD.
  • the term "about” is used herein to mean an amount that may differ somewhat from the given value, by an amount that would not be expected to significantly affect activity or outcome as appreciated by one of skill in the art, for example, a variance of from 1-10% from the given value.
  • exosomes comprising a lysosomal protein, and/or nucleic acid encoding the protein, for example, to treat a lysosomal storage disease, may be used in conjunction with (at different times or simultaneously, either in combination or separately) one or more additional therapies to facilitate treatment, including but not limited to; protein-specific modifications (i.e., GILT-tagged or carbohydrate re-modelled (mannose-6-phosphate enriched)), exercise, molecular chaperone compounds, or substrate reduction therapies (e.g. Miglustat).
  • protein-specific modifications i.e., GILT-tagged or carbohydrate re-modelled (mannose-6-phosphate enriched)
  • exercise e.g., exercise, molecular chaperone compounds
  • substrate reduction therapies e.g. Miglustat
  • a molecular chaperone may be included within the exosome or administered separately up to 2 hours prior to exosome infusion to increase stability of the lysosomal protein and further enhance ERT stability when exosomes are delivering protein.
  • Examples of such chaperones include, but are not limited to, endogenous proteins such as Hsc70, Hsp40, Hsp70, Hsp90, Hip, and BAG-1; chemical compounds such as the imino sugars, N-butyl-deoxynojirimycin, 1- deoxygalactonojirimycin; and like compounds.
  • the modified and unmodified exosomes of the present invention provide many advantages over current treatment methods such as enzyme replacement therapy.
  • exosomes exhibit greater uptake than other delivery means into tissues and into organelles such as lysosomes given that exosomes are naturally part of the lysosomal/endosomal recycling pathway, and thus, represent a physiological treatment method.
  • the present exosomes provide improved stability and protection from degradation and denaturation to proteins/enzymes or nucleic acid that they deliver. This in turn results in higher protein or nucleic acid delivery rates.
  • Exosomes can also cross the blood-brain barrier, allowing for the delivery of protein or nucleic acid to the central nervous system and, thereby being useful to treat conditions that involve the brain, e.g. LSDs such as neuronal ceroid-Lipofuscinosis (Batten disease), Tay-Sachs disease, metachromatic leukodystrophy and Niemann-Pick C.
  • LSDs such as neuronal ceroid-Lipofuscinosis (Batten disease), Tay-Sachs disease, metachromatic leukodystrophy and Niemann-Pick C.
  • the use of exosomes to deliver protein/nucleic acid results in a minimal immune reaction to the protein or nucleic acid being delivered because exosomes may be obtained from cells that do not induce any significant immunogenic response or which are not toxic (e.g. exosomes from immature dendritic cells).
  • the present exosomes may be tailored to incorporate a targeting sequence that results in enhanced recognition and
  • CHO cells transfected with GAA-pGEX vector were lysed and CHO cell lysate was cleared using ultra-performance resins for GST-tagged fusion protein purification (GE Healthcare Life Sciences). Over 80% of the recombinant protein was eluted after 3 washes. Elution #1 and Elution #2 were combined to obtain a high yield of protein. GST tag was removed from active GAA using PreScission Protease (GE Healthcare Life Sciences).
  • GAA cDNA was sub-cloned and amplified from skeletal muscle (from mouse and human). Using conventional PCR, start codon (ATG) and Kozak sequence (GCCACC) were introduced. This cDNA was then cloned into the pMRNA xp plasmid using EcoRI and BamHI restriction enzyme sites followed by transformation of competent E. coli DH5alpha line (Life Technologies). The colony containing the vector with positive insert was amplified. The vector was isolated from these colonies (Qiagen) and T7 RNA polymerase-based in vitro transcription reaction was carried out.
  • An anti-reverse cap analog (ARCA), modified nucleotides (5-Methylcytidine-5'-Triphosphate and Pseudouridine-5'- Triphosphate) and poly-A tail were incorporated into the mRNAs to enhance the stability and to reduce the immune response of host cells.
  • DNase I digest and phosphatase treatment was carried out to remove any DNA contamination and to remove the 5' triphosphates at the end of the RNA to further reduce innate immune responses in mammalian cells, respectively.
  • the clean-up spin columns were used to recover GAA mRNA for downstream encapsulation in engineered exosomes.
  • Exosomes expressing CD9-LamplTS fusion plasmid were then prepared for introduction of GAA (protein) into lysosomes.
  • Dendritic cells were isolated from mouse bone marrow progenitor cells and from human peripheral blood mononuclear cells (collected using Ficoll gradient separation of human blood). Briefly, femur and tibia were carefully harvested from mice and were flushed with HBSS media to collect bone marrow progenitor cells.
  • the bone marrow progenitor cells were cultured in GlutaMAX-DMEM media (Life Technologies) containing 10% FBS, lmM sodium pyruvate, 0.5% penicillin-streptomycin, and mouse recombinant granulocyte/macrophage colony-stimulating factor (R&D Systems).
  • GlutaMAX-DMEM media Life Technologies
  • penicillin-streptomycin penicillin-streptomycin
  • mouse recombinant granulocyte/macrophage colony-stimulating factor R&D Systems.
  • blood was collected in EDTA-lavender tubes followed by dilution of blood with 4x PBS buffer (pH 7.2 and 2 mM EDTA). 40 mL of diluted cell suspension was carefully layered over 20 mL of Ficoll gradient. The gradient was centrifuged at 400x g for 60 minutes followed by collection of the interphase layer containing the mononuclear cells.
  • the mononuclear cells were cultured in FMDM media (BD Biosciences) containing 10% FBS, 1% glutamine, 0.5% penicillin-streptomycin, and human recombinant granulocyte/macrophage colony-stimulating factor (R&D Systems). Both human and mouse dendritic cells were further purified using EasySepTM Mouse and Human Pan-DC Enrichment Kit (Stem Cell Technologies). Dendritic cells were then cultured with the aforementioned media (GlutaMAX-DMEM media for mouse DC and FMDB media for human DC).
  • the CD9-LamplTS fusion plasmid was made using Gateway® technology and vectors (Life Technologies) with amplified skeletal muscle (mouse and human) cDNA that corresponds to LAMP1 protein lysosomal targeting sequence (for human: MAAPGSARRPLLLLLLLLLLGLMHCASA (SEQ ID NO: 29); for mouse: MAAPGARRPL LLLLLAGLAHGASALFEVKN (SEQ ID NO: 30)) + first 10 amino acids of mature LAMP1 protein (for human: AMFMVKNGNG (SEQ ID NO: 31); for mouse: LFEVKNNGTT (SEQ ID NO: 32)).
  • the CD9 cDNA exosome marker (for mouse: NM_007657; for human: NM_001769.3) was amplified from mouse and human dendritic cell cDNA).
  • the lysosomal targeting sequence and the exosome marker were linked via PCR, and then were incorporated into a mammalian expression vector.
  • the dendritic cells were washed and combined with fresh growth media (GlutaMAX-DMEM media for mouse DC and EVIDB media for human DC, both media containing pre-spun exosome depleted FBS).
  • the dendritic cells were then grown to about 80% confluency in alpha minimum essential medium supplemented with ribonucleosides, deoxyribonucleosides, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng/mL murine GM-CSF, and 20% fetal bovine serum.
  • alpha minimum essential medium supplemented with ribonucleosides, deoxyribonucleosides, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng/mL murine GM-CSF, and 20% fetal bovine serum.
  • conditioned media collection cells were washed twice with sterile PBS (pH 7.4, Life Technologies) and the aforementioned media (with exosome-depleted fetal bovine serum) was added.
  • Conditioned media from human and mouse immature dendritic cell culture was collected after 48 hours.
  • the media (10 mL) was spun at 2,000x g for 15 min at 4°C to remove any cellular debris. This is followed by an optional 2000x g spin for 60 min at 4°C to further remove any contaminating non-adherent cells.
  • the supernatant was then spun at 14,000x g for 60 min at 4°C.
  • the resulting supernatant was spun at 50,000x g for 60 min at 4°C.
  • the supernatant was then filtered through a 40 ⁇ filter, followed by filtration through a 0.22 ⁇ syringe filter (twice). The supernatant was then carefully transferred into ultracentrifuge tubes and diluted with an equal amount of sterile PBS (pH 7.4, Life Technologies).
  • the exosomal pellet-containing fraction at the gradient interface was isolated carefully, diluted in 50 mL of sterile PBS (pH 7.4, Life Technologies), followed by a final spin for 90 minutes at 100,000x-170,000x g at 4°C to obtain purified exosomes.
  • the resulting exosomal pellet was resuspended in sterile PBS or sterile 0.9% saline for downstream use.
  • Exosomal fraction purity was confirmed by sizing using a Beckman DelsaMax dynamic light scattering analyzer showing minimal contamination outside of the 40-120 nm size range, and by immuno-gold labelling/Western blotting using the exosome membrane markers, CD9, CD63, TSG101 and ALIX.
  • Yield was about 1 x 10 9 particles around -100 nm in size. Using the PierceTM BCA protein quantification assay (Thermo Scientific), the yield of exosomes was estimated and found to be between 10 - 15 ug of exosomes.
  • Electroporation mixture was prepared by carefully mixing CD9-LAMP1 -tagged
  • electroporation was carried out in 0.4 mm electroporation cuvettes at 400 mV and 125 ⁇ capacitance (pulse time 14 milliseconds (ms) for mRNA and 24 ms for protein) using Gene Pulse XCell electroporation system (BioRad).
  • exosomes were resuspended in 20 mL of 0.9% saline solution followed by ultracentrifugation for 2 hours at 170,000x g at 4°C.
  • GAA mRNA or protein
  • GAA-loaded exosomes CD9-LAMP1 tagged exosomes carrying GAA protein or non-tagged exosomes carrying GAA mRNA
  • exosomes were loaded with GAA mRNA or protein using cationic lipid-based transfection reagents (Lipofectamine® MessengerMAXTM Transfection Reagent, Life Technologies). After transfection, exosomes were spun for 2 hours at 170,000x g at 4°C followed by re-suspension in 5% (wt/vol) glucose in 0.9% sterile saline solution.
  • GAA mRNA or GAA protein-loaded exosomes rescue GAA deficiency in primary fibroblasts and myotubes
  • GAA heterozygous breeding mouse pairs (GAA + ) were obtained from GAA + .
  • GAA knock-outs mice were genotyped at 1 month of age using a standard genotyping kit (REDExtract-N-Amp Tissue PCR Kit; Sigma Aldrich). Homozygous GAA knock-out mutants exhibited significant deficits in front-limb muscle strength as early as 1.5 months of age.
  • all animals were housed three to five per cage in a 12-h light/dark cycle and were fed ad libitum (Harlan-Teklad 8640 22/5 rodent diet) after weaning. The study was approved by the McMaster University Animal Research and Ethics Board under the global Animal Utilization Protocol # 12-03-09, and the experimental protocol strictly followed guidelines put forth by Canadian Council of Animal Care.
  • GAA activity and GAA-/- (GAA knock-out mice, model of Pompe Disease) were harvested using standard isolation techniques.
  • Myoblasts were differentiated into myotubes for exosome- treatment experiments. Fibroblasts and myotubes were treated with recombinant murine GAA protein (40 mg/kg) or 10 ug (total exosomal protein) of empty murine exosomes (not loaded with GAA mRNA or protein), exosomal GAA protein (modified exosomes loaded with murine GAA protein, 40 mg/kg of recombinant GAA in 10 ug of total exosomal protein), or exosomal GAA mRNA (mRNA dose equivalent to delivery of 40 mg/kg GAA, -100-150 ng mRNA, in 10 ug of total exosomal proteins in which exosomes were unmodified) for 48 hours in pre-spun growth media devoid of bovine microvesicles and exosomes.
  • fibroblasts and myotubes were prepared by homogenizing cells (fibroblasts and myotubes) in 200 [iL mannitol buffer (70 mM sucrose, 220 mM mannitol, 10 mM HEPES, 1 mM EGTA, protease inhibitor mixture (Complete Tablets, Roche), pH 7.4).
  • mannitol buffer 70 mM sucrose, 220 mM mannitol, 10 mM HEPES, 1 mM EGTA, protease inhibitor mixture (Complete Tablets, Roche), pH 7.4
  • Primary fibroblasts ( Figure 1) and primary myotubes ( Figure 2) show partial to complete rescue of GAA activity when treated with exosomal GAA protein (modified exosomes) or mRNA (unmodified exosomes), respectively, which is 2 to 8-fold higher than GAA rescue achieved by treatment with naked GAA (representative of conventional enzyme replacement therapy (ERT)).
  • exosomal GAA protein modified exosomes
  • mRNA unmodified exosomes
  • GAA mRNA or GAA protein-loaded exosomes prevent GAA deficiency in primary dermal fibroblasts isolated from Pompe patients
  • Fibroblasts treated with naked GAA protein and exosomal GAA protein were labeled with DAPI (nuclear marker), phalloidin (cellular cytoskeletal marker), and carboxyfluorescein succinimidyl diacetate ester (non-specific labeling of protein GAA) and subsequently imaged.
  • DAPI nuclear marker
  • phalloidin cellular cytoskeletal marker
  • carboxyfluorescein succinimidyl diacetate ester non-specific labeling of protein GAA
  • mice Dosage of naked GAA protein and GAA protein-loaded exosome was 40 mg of GAA protein/kg, given intravenously in 0.9% sterile saline. The amount of GAA loaded in exosomes was measured against a standard curve of GAA activity using purified recombinant GAA. The mice were tested for paw-grip endurance test, grip strength, and motor function at the beginning and end of the study (24 hours prior to harvesting tissue). Skeletal muscle (quadriceps, tibialis anterior, EDL, soleus, and diaphragm), heart, and brain were harvested from mice in all treatment groups.
  • [001 16] GAA enzyme activity A standard fluorometric enzyme assay as described above was used to determine acid a-glucosidase activity in cellular lysates prepared from fibroblasts and myotubes.
  • Total glycogen content in the tissue samples was determined using a fluorometric assay modified for a monochromator-based microplate detection system (Tecan Safire 2, Tecan Group Ltd, Mannedorf, Switzerland). In brief, 10 iL of glucose standards (2.5 ⁇ -600 ⁇ ) and neutralized samples was loaded in triplicate onto a 96 well black plate.
  • assay buffer 50 mM Tris, 1 mM MgCl 2 , 0.5 mM DTT, 300 ⁇ ATP, 50 ⁇ NADP, 0.02 U/mL glucose-6-P dehydrogenase in DW, pH 8.1
  • assay buffer 50 mM Tris, 1 mM MgCl 2 , 0.5 mM DTT, 300 ⁇ ATP, 50 ⁇ NADP, 0.02 U/mL glucose-6-P dehydrogenase in DW, pH 8.1
  • Net fluorescence values were obtained by subtracting the baseline read (and blanks) from the final read and unadjusted glycogen concentrations were calculated using the regression formula from the standard curve. Total glycogen concentrations were adjusted by an extraction dilution factor (dry tissue weight and volume acid) and reported as mmol/mg dry tissue weight.
  • GAA protein-loaded exosome therapy enhances strength and motor control, increases muscle mass, and reduces pathogenic cardiac hypertrophy vs. conventional naked GAA ERT in GAA KO mice.
  • 7-week treatment of GAA KO mice with GAA protein-loaded exosomes resulted in reduced body weight (Figure 5), and increased grip endurance time (A), grip strength (B), and rotarod fall time (C)( Figure 6) in GAA KO mice when compared to GAA KO mice treated with naked GAA protein (conventional enzyme replacement therapy (ERT)).
  • GAA KO mice treated with GAA protein-loaded exosomes showed improvements in predominantly slow- or fast-twitch fiber skeletal muscle mass (soleus and EDL, respectively) along with an increase in mass of mixed fiber-type muscles (quadriceps, gastrocnemius and tibialis anterior (TA)) in comparison to GAA KO mice treated with conventional naked GAA protein ERT ( Figures 7 and 8).
  • a significant reduction in heart mass and brain mass (to normal levels) of GAA KO mice treated with GAA protein-loaded exosomes was also observed when compared to GAA KO mice treated with conventional naked GAA protein ERT ( Figure 9).
  • GAA protein-loaded exosome therapy restores GAA activity in all tissue tested vs.
  • GAA protein-loaded exosome therapy clears pathological accumulation of glycogen. Additionally, 7-week treatment of GAA protein-loaded exosomes in GAA KO mice reduced pathological build-up of glycogen in skeletal muscle and heart to a greater degree than that conventional naked GAA ERT in GAA KO mice ( Figure 11). Similarly, clearance of total glycogen in the brain following treatment with GAA protein-loaded exosomes, but not following treatment with GAA ERT, was observed, depicting that GAA-packaged exosomes do cross the blood-brain barrier and have functional consequences (Figure 11). Inability to cross the blood- brain barrier is a major limitation of conventional intravenous administration of ERT.
  • GAA mRNA-loaded exosomes therapy restores GAA activity and GAA mRNA in all tissue tested to wild-type mice levels
  • GAA mRNA-loaded unmodified exosomes (no including a lysosomal targeting fusion product). Exosomes were loaded with an mRNA dose equivalent to delivery of 40 mg/kg GAA, -100-150 ng mRNA, in 10 ug of total exosomal proteins, isolated and prepared as described in Example 1, was conducted.
  • mice were harvested four days after the last intravenous bolus of GAA mRNA-loaded exosomes and the GAA activity was maintained in all tissues tested.
  • GAA mRNA expression was maintained in skeletal muscle ⁇ quadriceps femoris) and brain in GAA KO mice treated with GAA mRNA-loaded exosomes four days after the last injection ( Figure 14).
  • This result shows that there are physiological alterations in mRNA stabilizing proteins and/or the miRNA network in GAA KO mice only (where GAA protein activity is negligible) that prevents degradation of the GAA mRNA delivered, compared to WT mice where mRNA levels do not increase significantly (since they have optimal GAA activity).
  • tissues from WT mice treated with GAA mRNA-loaded exosomes did not show an up-regulation/overexpression of GAA activity indicating that there are inherent pathways that protect against an abnormal increase in GAA activity above physiological levels. From the perspective of treatment safety, this indicates the potential to avoid non-specific effects of very high levels of GAA activity (such as levels achieved by strategies like AAV-mediated GAA induction) using exosomal protein or nucleic acid delivery.
  • GAA mRNA-loaded exosomes therapy normalized neuromuscular function in GAA KO mice to wildtype mice levels
  • Exosome treatment was with 10 ug of exosomes (unmodified with a lysosomal tag) in 0.9% sterile saline loaded with either NPCl mRNA (100 ng) or rNPCl protein (100 ug). Isolation of exosomes and loading with NPCl mRNA and protein was conducted using methods similar to those described for GAA in Example 1.
  • the NPCl homozygous mice are known to have reduced levels of myelin in the cerebellum, and the astrocytes and microglia in these mice proliferate and occupy areas of neuronal loss or degeneration. Weight loss is accompanied by a progressive motor coordination deficit, or ataxia, at least as early as postnatal day 45 (P45). The lifespan of homozygous animals is reduced to a mean of 76 days.
  • NPC1 + + were treated (intravenously) with either empty exosomes (10 ug of total exosomal protein) or exosomes loaded with mouse npcl mRNA (3.33 ng of mRNA per g of mouse in 10 ug of total exosomal protein) in 0.9% saline solution per week for 5 weeks.
  • mice were tested for paw-grip endurance, grip strength, and motor function at the beginning and end of the study (24 hours prior to harvesting tissue).
  • the NPCl homozygous mouse treated with NPCl mRNA-loaded exosomes exhibited many fold higher values for paw-grip endurance, grip strength and motor function as compared to an NPCl homozygous mice treated with empty exosomes ( Figure 18).
  • the untreated NPC " mouse was so ataxic that the animal facility staff had to provide gel and food on the floor of the cage due to complete inability to reach water and food, whilst the treated mouse was indistinguishable from wild type mice.
  • Gaucher disease is a genetic disorder in which glucocerebroside (a sphingolipid) accumulates in cells and certain organs. It is characterized by bruising, fatigue, anemia, low blood platelet count and enlargement of the liver and spleen, and is caused by hereditary deficiency of the enzyme, ⁇ -glucocerebrosidase or glucosylceramidase (GBA). The following experiment is conducted to determine if this enzyme could be delivered to cells using exosomes.
  • glucocerebroside a sphingolipid
  • Gba mRNA cloned from the human genome (NCBI Accession #: NM_000157.3) empty exosomes (not loaded with Gba mRNA), or exosomal Gba mRNA (bioengineered exosomes loaded with human Gba mRNA cloned from human genome) for 48 hours in pre-spun growth media devoid of bovine microvesicles and exosomes.
  • exosomes (10 ug of total exosomal protein in 0.9% sterile saline solution) loaded with Gba mRNA (100 ng) (exosome isolation and loading is conducted as described in Example 1 for exosome isolation and GAA exosome loading). Exosomes may be modified with a fusion lysosomal targeting product.
  • Gba activity of treated and untreated dermal fibroblasts may be determined as previously described (Pasmanik-Chor, M, et al, Biochem J, 317:81-88, 1996). Primary fibroblasts from Gaucher patients are expected to show partial to complete rescue of GBA activity when treated with exosomal Gba mRNA.
  • the foregoing demonstrates for the first time that mRNA and protein can be delivered using the present bioengineered exosomes to completely correct a genetic defect in vivo in which there is a protein/enzyme deficiency.
  • the present method results in upregulation of a target protein in vivo by at least about 10%, and preferably by at least about 50%) or more, including upregulation of the protein to normal, wild-type levels, and further to effect recovery of normal function, activity and anatomy in a mammal.
  • the present exosome-mediated therapy can effectively treat three different lysosomal storage diseases that collectively represent the main tissues that are variably affected in all lysosomal storage diseases (e.g. brain, muscle, heart, liver, spleen, bone marrow and bone) and that the cellular pathophysiology of each lysosomal storage disease is the same, as one skilled in the art will appreciate, the present exosome-mediated therapy is applicable to all genetic disorders that affect the lysosome.
  • lysosomal storage diseases e.g. brain, muscle, heart, liver, spleen, bone marrow and bone

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Abstract

On décrit des exosomes génétiquement modifiés pour incorporer une protéine lysosomale fonctionnelle et/ou un acide nucléique codant pour la protéine lysosomale fonctionnelle, ainsi que l'utilisation desdits exosomes génétiquement modifiés pour traiter une maladie de stockage lysosomal.
PCT/CA2015/050959 2014-09-26 2015-09-25 Exosomes utiles pour traiter une maladie de stockage lysosomal WO2016044947A1 (fr)

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GB2552460A (en) * 2016-07-11 2018-01-31 Evox Therapeutics Ltd CPP-Mediated EV Loading
CN108473973A (zh) * 2016-09-30 2018-08-31 赛尔莱克斯生命科学公司 包含负载蛋白的外泌体的组合物以及制备和递送该组合物的方法
WO2019092287A1 (fr) * 2017-11-13 2019-05-16 Evox Therapeutics Ltd Vésicules extracellulaires modifiées par des protéines
WO2019243574A1 (fr) * 2018-06-22 2019-12-26 Evox Therapeutics Ltd Thérapie génique combinatoire
WO2020023594A1 (fr) * 2018-07-24 2020-01-30 Mayo Foundation For Medical Education And Research Compositions et procédés impliquant la transformation de vésicules extracellulaires
WO2020106099A1 (fr) * 2018-11-22 2020-05-28 (주)프로스테믹스 Exosome et utilisations diverses correspondantes
WO2020152298A1 (fr) 2019-01-24 2020-07-30 Fundació Hospital Universitari Vall D'hebron - Institut De Recerca Procédé de production d'enzymes
EP3648777A4 (fr) * 2017-07-06 2021-04-21 Children's National Medical Center Exosomes et procédés d'utilisation
WO2021183596A1 (fr) * 2020-03-10 2021-09-16 University Of Cincinnati Matériels et méthodes pour le traitement de la maladie de gaucher
US11208458B2 (en) 2017-06-07 2021-12-28 Regeneron Pharmaceuticals, Inc. Compositions and methods for internalizing enzymes
US11872193B2 (en) 2015-05-04 2024-01-16 Ilias Biologics Inc. Compositions containing protein loaded exosome and methods for preparing and delivering the same

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US20010031741A1 (en) * 2000-02-17 2001-10-18 Robin Ziegler Methods for treatment of lysosomal storage diseases
WO2007126386A1 (fr) * 2006-05-03 2007-11-08 Loetvall Jan Olof Transfert d'exosomes d'acides nucléiques à des cellules
WO2010119256A1 (fr) * 2009-04-17 2010-10-21 Isis Innovation Limited Composition pour la distribution de matériel génétique

Cited By (19)

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US11872193B2 (en) 2015-05-04 2024-01-16 Ilias Biologics Inc. Compositions containing protein loaded exosome and methods for preparing and delivering the same
EP3782639A1 (fr) * 2015-12-08 2021-02-24 Regeneron Pharmaceuticals, Inc. Compositions et méthodes pour l'internalisation d'enzymes
WO2017100467A3 (fr) * 2015-12-08 2017-07-20 Regeneron Pharmaceuticals, Inc. Compositions et méthodes permettant l'internalisation d'enzymes
EP4074331A1 (fr) * 2015-12-08 2022-10-19 Regeneron Pharmaceuticals, Inc. Compositions et méthodes pour l'internalisation d'enzymes
GB2552460A (en) * 2016-07-11 2018-01-31 Evox Therapeutics Ltd CPP-Mediated EV Loading
US11298319B2 (en) 2016-07-11 2022-04-12 Evox Therapeutics Ltd Cell penetrating peptide (CPP)-mediated EV loading
CN108473973A (zh) * 2016-09-30 2018-08-31 赛尔莱克斯生命科学公司 包含负载蛋白的外泌体的组合物以及制备和递送该组合物的方法
US11208458B2 (en) 2017-06-07 2021-12-28 Regeneron Pharmaceuticals, Inc. Compositions and methods for internalizing enzymes
EP3648777A4 (fr) * 2017-07-06 2021-04-21 Children's National Medical Center Exosomes et procédés d'utilisation
WO2019092287A1 (fr) * 2017-11-13 2019-05-16 Evox Therapeutics Ltd Vésicules extracellulaires modifiées par des protéines
JP2021502105A (ja) * 2017-11-13 2021-01-28 エヴォックス・セラピューティクス・リミテッド タンパク質操作された細胞外小胞
CN111601607A (zh) * 2017-11-13 2020-08-28 医福斯治疗有限公司 蛋白质工程化的细胞外囊泡
WO2019243574A1 (fr) * 2018-06-22 2019-12-26 Evox Therapeutics Ltd Thérapie génique combinatoire
WO2020023594A1 (fr) * 2018-07-24 2020-01-30 Mayo Foundation For Medical Education And Research Compositions et procédés impliquant la transformation de vésicules extracellulaires
KR102233530B1 (ko) 2018-11-22 2021-03-31 (주)프로스테믹스 엑소좀 및 이의 다양한 용도
KR20200060637A (ko) * 2018-11-22 2020-06-01 (주)프로스테믹스 엑소좀 및 이의 다양한 용도
WO2020106099A1 (fr) * 2018-11-22 2020-05-28 (주)프로스테믹스 Exosome et utilisations diverses correspondantes
WO2020152298A1 (fr) 2019-01-24 2020-07-30 Fundació Hospital Universitari Vall D'hebron - Institut De Recerca Procédé de production d'enzymes
WO2021183596A1 (fr) * 2020-03-10 2021-09-16 University Of Cincinnati Matériels et méthodes pour le traitement de la maladie de gaucher

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