EXOSOMES USEFUL TO TREAT LYSOSOMAL STORAGE DISEASE Field of the Invention

[0001] The present invention generally relates to exosomes, and more particularly, to the use of exosomes to treat a lysosomal storage disease.

Background of the Invention

[0002] Lysosomes are cellular organelles that function as a cellular waste disposal mechanism to remove unused, damaged, or excessive macro-molecules. There are at least fifty lysosomal enzymes that are known to function in the acidic environment of the lysosome (pH approximately 4.0-5.0) to break down cellular macro-molecules including; lipids, carbohydrates, glycolipids, proteins, nucleic acids and organelles such as mitochondria (mitophagy). In addition to the removal of accumulated cellular debris, 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. Collectively, 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. Once 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. The distinction between the MVBs and some late endosomes remains unclear; however, lysosomes are usually characterized by the presence of a membrane protein such as lysosomal-associated membrane protein (LAMP), e.g. LAMPl or 2, and intra-lysosomal enzymes such as cathepsin D. [0003] Lysosomal storage diseases (LSDs) are disorders resulting from gene mutations that lead to a defective, non-functional lysosomal protein (enzyme). There are over two dozen known genetic diseases that affect the lysosome. In general, 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.

[0004] One example of 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.

[0005] Until recently, therapy for LSDs was designed to treat the clinical manifestations of the disease (i.e. anti-seizure drugs for epilepsy, analgesics for pain, bracing for skeletal deformities, etc.), rather than the underlying cause of the LSD. More recently, two therapeutic strategies have been employed to specifically address and alleviate the biological deficiency that leads to LSDs. The first therapy is the direct replacement of the defective enzyme through intravenous infusion, called enzyme replacement therapy (ERT). The second therapy is the use of molecular chaperones that can enhance the residual activity of a dysfunctional enzyme and/or stabilize ERT proteins. 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. [0006] There are a variety of issues that can arise with enzyme replacement therapy that limit its effectiveness. Firstly, the pH of the lysosomes is quite low whereas blood is usually at a pH of 7.4. Consequently, 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. It is known that 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). 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. Ideally, a carrier system is needed that can reduce immunogenicity of the infused ERT.

[0007] One of the greatest limitations of conventional intravenous administration of ERT is that 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. Consequently, it would be desirable to have a method to deliver disease-specific ERT or mRNA to the central nervous system via an intravenous, sub -cutaneous, or oral route as opposed to intra-cerebral or intraventricular delivery method. [0008] Accordingly, it would be desirable to develop a novel treatment for pathological conditions resulting from a deficiency in the function of a particular protein such as a lysosomal protein.

Summary of the Invention

[0009] It has now been determined that 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.

[0010] Thus, in one aspect of the invention, exosomes that are genetically modified to incorporate a functional lysosomal protein and/or nucleic acid encoding a functional lysosomal protein.

[0011] In another aspect, a method of increasing the amount of a lysosomal protein in lysosomes in a mammal is provided, 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.

[0012] In another aspect, a method of increasing the activity of a target protein in a mammal is provided, comprising administering to the mammal exosomes which are genetically modified to incorporate a functional protein and/or nucleic acid encoding a functional protein.

[0013] In another aspect, a method of treating a pathological condition in a mammal resulting from the deficiency of a protein is provided comprising administering to the mammal exosomes genetically engineered to incorporate the protein or nucleic acid encoding the protein.

[0014] In a further aspect, a method of treating a lysosomal storage disease in a mammal is provided 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.

[0015] Additional aspects of the invention are include aspects and variations set forth in the following lettered paragraphs: [0016] Al . An exosome produced by a process that comprises: (a) isolating exosomes from a biological sample from an organism or from a conditioned medium from a cultured cell; and (b) introducing a modification into the exosome selected from the group consisting of:

(i) at least one functional lysosomal protein or precursor thereof;

(ii) at least one nucleic acid comprising a nucleotide sequence that encodes the functional lysosomal protein or precursor thereof;

(iii) at least one fusion product comprising a lysosome targeting sequence linked to an exosomal membrane marker;

(iv) at least one nucleic acid comprising a nucleotide sequence that encodes the fusion product; and

(v) two or more of (i), (ii,) (iii), and (iv).

[0017] A2. The exosome according to paragraph Al, wherein the isolating includes at least one density gradient centrifugation step ideally using Percoll or other colloidal silica product.

[0018] A2.1 The exosome according to paragraph Al or A2, wherein the isolating removes vesicles that are greater than 120 nm in diameter.

[0019] A3. The exosome according to paragraph Al or A2 or A2.1, wherein the biological sample is from a mammal, or the cell is from a mammal or a mammalian cell line.

[0020] A4. The exosome according to any one of paragraphs Al to A3, wherein the isolating removes vesicles and cellular debris less than 20 nm in diameter.

[0021] A5. An exosome that comprises a modification selected from the group consisting of:

(i) at least one functional lysosomal protein or precursor thereof; (ii) at least one nucleic acid comprising a nucleotide sequence that encodes the functional lysosomal protein or precursor thereof;

(iii) at least one fusion product comprising a lysosome targeting sequence linked to an exosomal membrane marker;

(iv) at least one nucleic acid comprising a nucleotide sequence that encodes the fusion product; and

(v) two or more of (i), (ii,) (iii), and (iv).

[0022] Bl . The exosome according to any of paragraphs Al - A5, having a diameter of 20-120 nm.

[0023] B2. The exosome according to any of paragraphs Al - A5, that comprises a functional lysosomal protein or precursor thereof, wherein the protein is present in a lumen of the exosome.

[0024] B3. The 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.

[0025] B3.1. The exosome according to paragraph B3, wherein the 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.

[0026] B4. The exosome according to paragraph B2 or B3 or B3.1, wherein the protein comprises one or more of the proteins set forth in Table 1.

[0027] B5. The exosome according to paragraph B4, wherein the protein is an enzyme.

[0028] B6. The exosome according to any one of paragraphs B2 - B3.1, wherein the protein is selected from the group consisting of alpha-D-mannosidase, N-aspartyl-beta- glucosaminidase, lysosomal acid lipase, cystinosin, lysosomal associated membrane protein-2, alpha-galactosidase A, acid ceramidase, alpha-fucosidase, cathepsin A, acid beta-glucosidase, beta-galactosidase, beta-hexosaminidase A, beta-hexosaminidase B, GlcNAc-1- phosphotransferase, beta-galactosylceramidase, lysosomal acid lipase, arylsulfatase A, alpha-L- iduronidase, iduronate-2-sulphatase, heparan sulphamidase, acetyl alpha-glucosaminidase, acetyl CoA: alpha-glucosaminide-N-acetyltransferase, N-acetyl glucosamine-6-sulfatase, N-acetyl galactosamine-6-sulfatase, hyaluronidase, acetyl galactosamine-4-sulphatase, beta- glucuronidase, alpha-N -acetyl neuraminidase, N-actiylglucosamine-1 -phosphotransferase, mucolipin-1, formylgly cine-generating enzyme, palmitoyl-protein thioesterase-1, tripeptidyl peptidase I, cysteine string protein, CLN3p, CLN5p, CLN6p, CLN7p, CLN8p, acid sphingomyelinase, PC 1, PC 2, acid alpha-glucosidase, cathepsin K, sialin, alpha-N- acetylgalactosaminidase, GM2 activator, lysosomal acid lipase.

[0029] B7. The 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.

[0030] B8. The 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.

[0031] B9. The 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.

[0032] 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 . The exosome of paragraph BIO, wherein the C-terminal sequence is selected from the group consisting of 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), GYRHV (SEQ ID NO: 11), DXXLL (SEQ ID NO: 12), SFHDD SDEDLL (SEQ ID NO: 14), EE SEERDDHLL (SEQ ID NO: 15), G YHDD SDEDLL (SEQ ID NO: 16), ASVSLLDDELM (SEQ ID NO: 17), AS SGLDDLDLL (SEQ ID NO: 18), VQNPSADRNLL (SEQ ID NO: 19), NALSWLDEELL (SEQ ID NO:20), TERERLL (SEQ ID NO: 21), SETERLL (SEQ ID NO: 22), TDRTPLL (SEQ ID NO: 23), EETQPLL (SEQ ID NO: 24), ITGFSDDVPMV (SEQ ID NO: 25), DERAPLI (SEQ ID NO: 26), NEQLPML (SEQ ID NO: 27) and DDQRDLI (SEQ ID NO: 28).

[0034] B12. The exosome according to any one of paragraphs B7 - B l l, wherein the fusion product is a fusion protein.

[0035] B13. The exosome according to paragraph B12, further wherein the fusion protein includes a peptide linker between the lysosome targeting sequence and the exosomal membrane marker.

[0036] B14. The exosome according to any one of paragraphs B7 - B13, wherein the lysosome targeting sequence is linked to the exosomal membrane marker with a hydrophobic linker comprising 4-5 hydrophobic amino acid moieties which are the same or different and selected from the group consisting of glycine, valine, leucine, isoleucine, methionine, alanine, proline, tryptophan and phenylalanine.

[0037] B15. The exosome according to any one of paragraphs B7-B13, wherein the fusion product includes a transmembrane domain and localizes in a membrane of the exosome.

[0038] CI . A composition comprising exosomes according to any one of paragraphs

AI - A5, and a pharmaceutically acceptable carrier.

[0039] C2. The composition according to paragraph CI, wherein the composition is substantially free of vesicles having a diameter less than 20 nm. [0040] C3. The composition according to paragraph CI or C2, wherein the composition is substantially free of vesicles having a diameter greater than 120mm.

[0041] C4. The composition according to any one of claims CI - C3, which exhibits a zeta potential having a magnitude of at least 30 mV, or at least 40 mV, or at least 50 mV, or at least 60 mV, or at least 70 mV, or at least 80 V.

[0042] C5. The 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.

[0043] 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.

[0044] D2. Use of an exosome according to any one of paragraphs Al - B15, or a composition according to any one of paragraphs CI- C5, for increasing the amount of a lysosomal protein in lysosomes in a mammal.

[0045] D3. 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.

[0046] D4. Use of an exosome according to any one of paragraphs Al - B15, or a composition according to any one of paragraphs CI- C5, for treating a lysosomal storage disease in a mammal.

[0047] D5. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human.

[0048] 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, MPS II - Hunter Syndrome, MPS IIIA - Sanfilippo Syndrome Type A, MPS IIIB - Sanfilippo Syndrome Type B, MPS IIIB - Sanfilippo Syndrome Type C, MPS IIIB - Sanfilippo Syndrome Type D, MPS IV - Morquio Type A, MPS IV - Morquio Type B, MPS IX - Hyaluronidase Deficiency, MPS VI - Maroteaux-Lamy, MPS VII - Sly Syndrome, Mucolipidosis I - Sialidosis, Mucolipidosis IIIC, Mucolipidosis Type IV, Multiple Sulfatase Deficiency, Neuronal Ceroid Lipofuscinosis Tl, Neuronal Ceroid Lipofuscinosis T2, Neuronal Ceroid Lipofuscinosis T3, Neuronal Ceroid Lipofuscinosis T4, Neuronal Ceroid Lipofuscinosis T5, Neuronal Ceroid Lipofuscinosis T6, Neuronal Ceroid Lipofuscinosis T7, Neuronal Ceroid Lipofuscinosis T8, Niemann-Pick Disease Type A, Niemann-Pick Disease Type B, Niemann-Pick Disease Type C, Pompe Disease, Pycnodysostosis, Salla Disease, Schindler Disease and Wolman Disease.

[0049] D7. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human and has a disease set forth in Table 1, and the exosome contains the corresponding protein in Table 1, or a nucleic acid encoding said protein.

[0050] The invention further includes numerous embodiments, aspects, and variations that will be apparent from the drawings, details description, and claims that follow.

[0051] These and other aspects of the invention will be described by reference to the following figures.

Brief Description of the Figures

[0052] 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. (A) Primary dermal fibroblasts isolated from GAA+/+ and GAA-/- mice (n = 3 per group) 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.

[0053] Figure 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. (A) Primary mononuclear cells were isolated from skeletal muscle of GAA+/+ and GAA-/- mice (n = 3 per group). Cells were pre-plated to obtain a pure population of myoblasts. Cells were differentiated for 5 days into myotubes followed by treatment 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.

[0054] 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.

[0055] 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. 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 glycogen content was measured. Mean ± SD of experiments independently repeated 3 times. *P < 0.05. Data were analyzed using an unpaired /-test. [0056] Figure 5 graphically illustrates that GAA protein-loaded exosomes therapy reduces body weight in GAA KO mice. Body weights of GAA KO mice (n = 5-7 per group) treated with saline (empty exosomes), naked recombinant GAA ERT (naked GAA), and GAA protein-loaded exosomes ERT for 7 weeks (once a week intravenously, corresponding to 40 mg/kg GAA). Data were analyzed using an unpaired t-test.

[0057] Figure 6 graphically illustrates that GAA protein-loaded exosome therapy increases strength and motor control vs. conventional naked GAA ERT in GAA KO mice.

(A) Paw-grip endurance test, (B) grip strength test, and (C) rotarod motor control test in GAA KO mice (n = 5-7 per group) treated with saline (empty exosomes), naked recombinant GAA ERT (naked GAA), and 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.

[0058] Figure 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) and soleus (slow- twitch muscle; red bar) mass in GAA KO mice (n = 5-7 per group) treated with saline (empty exosomes), naked recombinant GAA ERT (naked GAA), and 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 t-test.

[0059] Figure 8 graphically illustrates that GAA protein-loaded exosome therapy increases mixed fiber-type mass vs. conventional naked GAA ERT in GAA KO mice. (A)

Quadriceps, (B) gastrocnemius, and (C) tibialis anterior (TA) mass in GAA KO mice (n = 5-7 per group) treated with saline (empty exosomes), naked recombinant GAA ERT (naked GAA), and 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 t-test.

[0060] Figure 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 (B) brain mass in GAA KO mice (n = 5-7 per group) treated with saline (empty exosomes), naked recombinant GAA ERT (naked GAA), and 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.

[0061] Figure 10 graphically illustrates that GAA protein-loaded exosome therapy increases tissue GAA activity vs. conventional naked GAA ERT in GAA KO mice. (A) GAA activity in EDL, soleus, diaphragm, heart, and brain of GAA KO mice (n = 5-7 per group) treated with saline (empty exosomes) or conventional naked recombinant GAA ERT (naked GAA). (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.

[0062] Figure 11 graphically illustrates that GAA protein-loaded exosome therapy reduces tissue total glycogen content vs. conventional naked GAA ERT in GAA KO mice.

Total glycogen content (mmol glucosyl units/kg of dry tissue weight) in tibialis anterior (TA) muscle, heart, and brain of GAA KO mice (n = 5-7 per group) treated with saline (empty exosomes), conventional naked recombinant GAA ERT (naked GAA) and exosomal GAA. *P < 0.05. Data were analyzed using an unpaired /-test.

[0063] Figure 12 graphically illustrates that GAA mRNA-loaded exosome therapy increases skeletal muscle, diaphragm, and heart GAA activity in GAA KO mice. GAA activity is restored in fast- (EDL) and slow- (soleus) fiber-type skeletal muscle (A), and in the diaphragm and heart (B) in GAA KO mice (n = 5-6 per group) treated with exosomes loaded with GAA mRNA vs. GAA KO mice treated with empty exosomes (n = 5-6 per group). Littermate wildtype (WT) mice were used as controls for both treatments. *P < 0.05. Data were analyzed using an unpaired /-test.

[0064] Figure 13 graphically illustrates that GAA mRNA-loaded exosome therapy increases brain GAA activity in GAA KO mice. GAA activity is restored in the brain of GAA KO mice (n = 5-6 per group) treated with exosomes loaded with GAA mRNA vs. GAA KO mice treated with empty exosomes (n = 5-6 per group). Littermate wildtype (WT) mice were used as controls for both treatments. *P < 0.05. Data were analyzed using an unpaired /-test. [0065] Figure 14 graphically illustrates that GAA mRNA-loaded exosome therapy normalizes skeletal muscle and brain GAA gene expression in GAA KO mice. GAA mRNA expression is restored in the skeletal muscle (quadriceps) and brain of GAA KO mice (n = 5-6 per group) treated with exosomes loaded with GAA mRNA vs. GAA KO mice treated with empty exosomes (n = 5-6 per group). Littermate wildtype (WT) mice were used as controls for both treatments. *P < 0.05. Data were analyzed using an unpaired t-test.

[0066] Figure 15 graphically illustrates that GAA mRNA-loaded exosome therapy reduces pathological glycogen content in heart and skeletal muscle of GAA KO mice. Total glycogen content is normalized in skeletal muscle {quadriceps and tibialis anterior) and heart in GAA KO mice (n = 5-6 per group) treated with exosomes loaded with GAA mRNA vs. GAA KO mice treated with empty exosomes (n = 5-6 per group). Littermate wildtype (WT) mice were used as controls for both treatments. *P < 0.05. Data were analyzed using an unpaired t-test.

[0067] Figure 16 graphically illustrates that GAA mRNA-loaded exosome therapy increases grip endurance in GAA KO mice. GAA KO mice (n = 5-6 per group) treated with exosomes loaded with GAA mRNA vs. GAA KO mice treated with empty exosomes (n = 5-6 per group). Littermate wildtype (WT) mice were used as controls for both treatments. *P < 0.05. Data were analyzed using an unpaired t-test.

[0068] Figure 17 graphically illustrates that NPCl mRNA loaded exosome therapy rescues substrate accumulation in primary fibroblasts isolated from Niemann-Pick Disease Type C patient. Primary dermal fibroblasts isolated from three Niemann-Pick Disease Type C patients and three age/gender-matched controls, were treated with; media control, empty exosomes (exosome control), NPCl protein-loaded exosomes, or NPCl mRNA-loaded exosomes for 48 hours. 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.

[0069] Figure 18 graphically illustrates restoration of grip strength (A) and motor function (B = fall time on rotarod; C = fall speed on rotarod) in NPCl-/- homozygous mice treated with NPCl mRNA-loaded exosomes as compared to NPCl-/- homozygous mice treated with empty exosomes. Detailed Description of the Invention

[0070] Exosomes are provided which have been genetically engineered to incorporate a lysosomal protein, and/or nucleic acid encoding a lysosomal protein.

[0071] The term "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). As one of skill in the art will appreciate, 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. CD9, CD37, CD44, CD53, CD63, CD81, CD82 and CD151; targeting or adhesion markers such as integrins, ICAM-1, EpCAM and CD31; membrane fusion markers such as annexins, TSG101, ALIX; and other exosome transmembrane proteins such as Rab5b, HLA-G, HSP70, LAMP2 (lysosome-associated membrane protein) and LIMP (lysosomal integral membrane protein). Exosomes may also be obtained from a non-mammalian biological sample, including cultured non-mammalian cells. As the molecular machinery involved in exosome biogenesis is believed to be evolutionarily conserved, 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. As used herein, 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. The term "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. [0072] Exosomes may be obtained from the appropriate biological sample using a combination of isolation techniques, for example, centrifugation, filtration and ultracentrifugation methodologies. In one embodiment, 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.

[0073] Thus, 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. As one of skill in the art will appreciate, a suitable commercially available laboratory centrifuge, e.g. Thermo- Scientific™ or Cole-Parmer™, is employed to conduct this isolation step. To enhance exosome isolation, 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.

[0074] Following removal of cell debris, 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. In one embodiment, 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. [0075] 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. In one embodiment, the centrifugation step is conducted at 50,000x g for 1 hour. The resulting supernatant is separated from the pellet for further processing.

[0076] 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. Alternatively, filtration using 2 or more filters may be conducted, using 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.

[0077] The microfiltered supernatant (filtrate) 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. The exosomal solution is then subjected to ultracentrifugation to pellet exosomes and any remaining contaminating microvesicles (between 100-220 nm). 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-Scientific™ or Beckman™, 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.

[0078] Following ultracentrifugation, the re-suspended exosome-containing pellet is subjected to density gradient separation to separate contaminating microvesicles from exosomes based on their density. Various 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). Thus, examples of 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. Percoll™ 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.

[0079] Depending on the density gradient used, the re-suspended exosome pellet resulting from the density gradient separation may be ready for use. For example, if 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. If 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. subjected to one to three ultracentrifugation steps at a speed of 110,000- 170,000x g for 1-3 hours each at 4 °C, to yield an essentially pure exosome-containing pellet. As one of skill in the art will appreciate, 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. Alternatively, 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). [0080] 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. referred to herein as "essentially free" from cellular debris, apoptotic bodies and microvesicles having a diameter less than 20 or greater than 120 nm, and preferably free from particles having a diameter of less than 40 or greater than 120 nm, and which are biologically intact, e.g. not clumped or in aggregate form, and not sheared, leaky or otherwise damaged. 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. < -30 or > +30, and preferably, a magnitude of at least 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, or greater. The term "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. For exosomes, 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.

[0081] Moreover, high quantities of exosomes are achievable by the present isolation method, e.g. exosomes in an amount of about 100-2000 μg 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). Thus, solutions comprising exosomes at a concentration of at least about 5 μg/μL, and preferably at least about 10-25 μg/μL, 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.

[0082] 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. Thus, 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.

[0083] 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. The term "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. Similarly, 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® MessengerMAX™ Transfection Reagent, Lipofectamine® RNAiMAX Transfection Reagent, Lipofectamine® 3000 Transfection Reagent, or Lipofectamine® LTX Reagent with PLUS™ Reagent, may also be used. The amount of transfection reagent used may vary with the reagent, the sample and the cargo to be introduced. For example, using Lipofectamine® MessengerMAX™ Transfection Reagent, 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.

[0084] 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.

[0085] In another embodiment, 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.

[0086] Alternatively, 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. For the delivery of lysosomal proteins, 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. Examples of 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.

[0087] 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. Examples of 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. Thus, 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). Additionally, 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). Other 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).

[0088] To increase the efficiency of lysosome targeting, 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. Thus, 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.

[0089] Exosomes incorporating the lysosome-specific fusion product may be produced using recombinant technology. In this regard, 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. As one of skill in the art will appreciate, it is also possible to produce the fusion product using recombinant techniques, and then introduce the fusion product directly into exosome-producing cells using similar techniques, e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like. Following a sufficient period of time, exosomes generated by the exosome-producing cells, and including the fusion product, may be isolated as described.

[0090] 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.

[0091] 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. In one embodiment, 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. 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.

Table 1.

GM2 - Tay-Sachs disease beta-hexosaminidase A + B NM 000520.4

GM2 - Gangliosidosis, AB variant GM2 activator (GM2A) NM 000405.4

I-Cell Disease/Mucolipidosis II GlcNAc- 1 -phosphotransferase NM 024312.4

Krabbe Disease beta-galactosylceramidase NM 000153.3

Lysosomal acid lipase deficiency lysosomal acid lipase NM_000235

Metachromatic Leukodystrophy arylsulfatase A NM 000487.5

MPS I - Hurler Syndrome alpha-L-iduronidase NM 000203.4

MPS I - Scheie Syndrome alpha-L-iduronidase NM 000203.4

MPS I Hurler-Scheie Syndrome alpha-L-iduronidase NM 000203.4

MPS II - Hunter Syndrome iduronate-2-sulphatase NM 000202.6

MPS IIIA - Sanfilippo Syndrome Type A heparan sulphamidase NM 000199.3

MPS IIIB - Sanfilippo Syndrome Type B acetyl alpha-glucosaminidase NM 000263.3 acetyl CoA: alpha-glucosaminide-N-

MPS IIIC - Sanfilippo Syndrome Type C acetyltransferase NM 152419.2

MPS HID - Sanfilippo Syndrome Type D N-acetyl glucosamine-6-sulfatase NM 002076.3

MPS IV - Morquio Type A N-acetyl galactosamine-6-sulfatase NM 000512.4

MPS IV - Morquio Type B beta-galactosidase NM 000404.2

MPS IX - Hyaluronidase Deficiency Hyaluronidase NM 033159.3 acetyl galactosamine-4-sulphatase

MPS VI - Maroteaux-Lamy (arylsulphatase B) NM 000046.3

MPS VII - Sly Syndrome beta-glucuronidase NM 000181.3

Mucolipidosis I - Sialidosis alpha-N -acetyl neuraminidase NM 001304450.1

N-actrylglucosamine- 1 -

Mucolipidosis IIIC phosphotransferase NM 032520.4

Mucolipidosis Type IV mucolipin-1 NM 020533.2

Multiple Sulfatase Deficiency formylglycine-generating enzyme NM 001042469.1

Neuronal Ceroid Lipofuscinosis Tl palmitoyl -protein thioesterase-1 NM 000310.3

Neuronal Ceroid Lipofuscinosis T2 tripeptidyl peptidase I NM 001020382.2

Neuronal Ceroid Lipofuscinosis T3 CLN3p NM 001283889.1

Neuronal Ceroid Lipofuscinosis T4 cysteine string protein NM 025219.2

Neuronal Ceroid Lipofuscinosis T5 CLN5p NM 006493.2

Neuronal Ceroid Lipofuscinosis T6 CLN6p NM 017882.2

Neuronal Ceroid Lipofuscinosis T7 CLN7p NM 152778.2

Neuronal Ceroid Lipofuscinosis T8 CLN8p NM 018941.3

Niemann-Pick Disease Type A acid sphingomyelinase NM 000543.4

Niemann-Pick Disease Type B acid sphingomyelinase NM 000543.4 Niemann-Pick Disease Type C NPC 1/ NPC 2 NM 000271.4

Pompe Disease acid alpha-glucosidase NM 000152.3

Pycnodysostosis cathepsin K NM 000396.3

Salla Disease sialin (sialic acid transporter) NM 012434.4

Schindler Disease alpha-N-acetylgalactosaminidase NM 000262.2

Wolman Disease lysosomal acid lipase NM 000235.3

[0092] As one of skill in the art will appreciate, 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; and 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)), galactose, mannose, N-acetylglucosamine, and fucose, fusion products (e.g. with Fc peptide), and the like. 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.

[0093] 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. The expressions "pharmaceutically acceptable" or "physiologically acceptable" means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable for physiological use. As one of skill in the art will appreciate, the selected carrier will vary with intended utility of the exosome formulation. In one embodiment, exosomes are formulated for administration by infusion or injection, e.g. subcutaneously, intraperitoneally, intramuscularly or intravenously, and thus, are formulated as a suspension in 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. lactated or acetated Ringer's solution, phosphate buffered saline (PBS), TRIS (hydroxymethyl) aminomethane hydroxymethyl) aminomethane)-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS) and Gey's balanced salt solution (GBSS). [0094] In other embodiments, 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. For oral administration, 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.

[0095] 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. Thus, for use to treat such a pathological condition, 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. The term "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. In one embodiment, 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. For example, 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.

[0096] As will be appreciated by one of skill in the art, 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). With respect to molecular chaperones, the addition of 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.

[0097] The modified and unmodified exosomes of the present invention provide many advantages over current treatment methods such as enzyme replacement therapy. At the outset, 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. In addition, 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. Further, 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). Moreover, the present exosomes may be tailored to incorporate a targeting sequence that results in enhanced recognition and fusion with the target organelle, e.g. lysosomes, to result in increased specificity with respect to targeted protein/nucleic acid delivery.

[0098] Embodiments of the invention are described by reference to the following specific examples which are not to be construed as limiting.

Example 1 - Treatment of acid a-glucosidase deficiency

Production of Human and Mouse GAA niRNA and Protein

[0099] Human acid a-glucosidase (GAA - NCBI Reference Sequence: M_000152.3) and mouse GAA (NCBI Reference Sequence: NM_008064.3) cDNA from skeletal muscle were sub-cloned into mammalian vector (pGEX GST-fusion vector; GE Healthcare Life Sciences). The vector was maintained using competent E. coli DH5alpha line (Life Technologies). The pGEX mammalian vector was then transfected into Chinese Hamster Ovary Cells (CHO; ATCC Cat. CCL-661) for mass production of active GAA enzyme. To isolate recombinant GAA in its processed active form, 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).

[00100] To synthesize GAA mRNA, 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.

Production of Non-immunogenic Exosomes Expressing CD9-LamplTS Fusion Plasmid

[00101] Exosomes expressing CD9-LamplTS fusion plasmid were then prepared for introduction of GAA (protein) into lysosomes. Dendritic cells (DC) 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). For human dendritic cell isolation, 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 EasySep™ 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). Media was pre-spun at 170,000x g for 2 hours at 37 °C for 4 days to ensure that the subsequent exosome pellet would not be contaminated with bovine microvesicles and/or exogenous exosomes. [00102] To prepare unmodified exosomes (not tagged with a membrane fusion product), cells were grown to about 80% confluency before exosome collection as described below. To prepare modified exosomes (tagged with a membrane fusion product), on the third day, DC were transfected with mammalian expression CD9-LamplTS fusion plasmid 0.1-1 ug (depending on cell density) using Lipofectamine 3000 reagent (Life Technologies). 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. On the fifth day, 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).

[00103] 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. For 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 μπι filter, followed by filtration through a 0.22 μπι 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). This mixture was then subjected to ultracentrifugation at 100,000x-170,000x g for 2 hours at 4°C using a fixed-angle rotor. The resulting pellet was re- suspended in PBS and re-centrifuged at 100,000x-170,000x g for 2 hours at 4°C. The pellet was resuspended carefully with 25 mL of sterile PBS (pH 7.4, Life Technologies) and then added gently on top of 4 mL of 30%/70% Percoll™ gradient cushion (made with 0.22 μιη filter sterilized water) in an ultracentrifuge tube. This mixture was spun at 100,000x-170,000x g for 90 minutes at 4°C. With a syringe, 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.

[00104] Purified exosomes were suspended in a 100-140 [iL of pre-chilled electroporation buffer (1.5 mM potassium phosphate pH 7.2, 25 mM KCl, and 21% (vol/vol) OptiPrep for GAA mRNA electroporation of pre-engineered exosomes OR 1.5 mM potassium phosphate pH 7.2, 25 mM KCl, 250 mM trehalose, 1 mM inositol, and 21% (vol/vol) OptiPrep for GAA protein electroporation of pre-engineered exosomes). Exosomes were then counted and sized by NanoSight nanoparticle tracking analysis (NanoSight, Ltd.). Yield was about 1 x 10 ⁹ particles around -100 nm in size. Using the Pierce™ BCA protein quantification assay (Thermo Scientific), the yield of exosomes was estimated and found to be between 10 - 15 ug of exosomes.

Introduction of GAA mRNA or protein into exosomes

[00105] Electroporation mixture was prepared by carefully mixing CD9-LAMP1 -tagged

(modified) exosomes and GAA protein in 1 : 1 ratio in electroporation buffer. Similarly, electroporation mixture was prepared by mixing non-tagged (unmodified) exosomes and GAA mRNA in 1 : 1 ratio in electroporation buffer. Electroporation was carried out in 0.4 mm electroporation cuvettes at 400 mV and 125 μΤ capacitance (pulse time 14 milliseconds (ms) for mRNA and 24 ms for protein) using Gene Pulse XCell electroporation system (BioRad). After electroporation, exosomes were resuspended in 20 mL of 0.9% saline solution followed by ultracentrifugation for 2 hours at 170,000x g at 4°C. For in vitro and in vivo exosome administration, GAA (mRNA or protein)-loaded exosomes (CD9-LAMP1 tagged exosomes carrying GAA protein or non-tagged exosomes carrying GAA mRNA) were re-suspended in 5% (wt/vol) glucose in 0.9% saline solution. Alternatively, exosomes were loaded with GAA mRNA or protein using cationic lipid-based transfection reagents (Lipofectamine® MessengerMAX™ 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

[00106] Four GAA heterozygous breeding mouse pairs (GAA ⁺ ) were obtained from

Jackson Laboratories (Maine, USA) to generate homozygous GAA knock-outs (GAA ) and wild-type littermates (GAA ^{+ +} ). 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. During breeding, 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.

[00107] Primary fibroblasts and myotubes from GAA+/+ (wild-type mice with normal

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. [00108] A standard fluorometric enzyme assay, originally described by Reuser et al.

(Biochem Biophys Res Commun _^ 1978 Jun 29;82(4): 1176-82), was used to determine acid a- glucosidase (GAA; EC 3.2.1.20) activity of treated and untreated fibroblasts and myotubes. In brief, cellular lysates 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). Following BCA assay (Pierce) for colorimetric determination of protein concentration, 10 iL of each sample (in triplicate) was mixed with 20 iL of the artificial acid a-glucosidase substrate, 4-methyl-umbelliferyl a-d-gluco- pyranoside, in 0.2 M sodium acetate [NaAc] buffer, pH 3.9, heated to 65 °C in a 96 well black plate. Standards were prepared from a 5 mM 4-methylumbelliferone/50% ethanol stock by serial dilution in 0.2 M NaAc buffer (pH 3.9), loaded in 10 μΐ, triplicates, and mixed with 20 |iL 0.2 M NaAc buffer (pH 3.9). The samples were then incubated in the dark for 1 h at 37.5 °C and the reaction was terminated by adding 200 [iL of 0.5 M sodium carbonate (pH 10.7). The release of the product, 4-methylumbelliferone, from the substrate is proportional to acid a-glucosidase activity (nmol/mg protein/hr), and the resulting fluorescence was read at 360 nm excitation/460 nm emission with a monochromator-based microplate detection system (Tecan).

[00109] 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)).

GAA mRNA or GAA protein-loaded exosomes prevent GAA deficiency in primary dermal fibroblasts isolated from Pompe patients

[00110] Primary dermal fibroblasts (grown from skin biopsies used to diagnose Pompe disease) from three Pompe patients and three healthy age/gender-matched controls were treated with naked human GAA recombinant protein or empty exosomes (not loaded with GAA mRNA or protein), exosomal GAA protein (modified exosomes loaded with human GAA protein), or exosomal GAA mRNA (unmodified exosomes loaded with human GAA mRNA) for 48 hours in pre-spun growth media devoid of bovine microvesicles and exosomes. [00111] GAA activity of treated and untreated fibroblasts was determined as described above. Primary fibroblasts from Pompe patients show partial to complete rescue of GAA activity when treated with exosomal GAA protein or mRNA, respectively (Figure 3). The rescue in GAA activity using GAA-exosome therapy was about 2.5 to 6-fold higher than that achieved by treatment with naked GAA (conventional ERT).

[00112] This was further confirmed by a significant decrease in fibroblast total glycogen content (Figure 4). To determine glycogen content, cell lysate was treated with 30% KOH, boiled for 15 min, and stored on ice for 30 min. Glycogen was precipitated using 66% ethanol followed by centrifugation (5000x g for 10 min at 4°C). The resulting pellet was resuspended in 300 uL of 0.1 M acetic acid. Glycogen concentration was determined by examining release of glucose after digestion of 100 μΐ. of glycogen-acetic acid mix with 0.1 U Aspergillus niger amyloglucosidase (Sigma) at 55°C for 30 min. The glucose concentration after hydrolysis is proportional to glucose reaction with ATP to form glucose-6-phosphate which converts NADP ⁺ into NADPH, spectrophotometrically determined at 340 nm.

[00113] 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. GAA rescue using exosomal delivery of GAA mRNA and protein was shown to be due to more efficient uptake thereof as compared to the uptake of naked GAA protein.

Example 2 - Treatment of GAA deficiency in vivo with Protein-loaded Exosomes Methods

[00114] Breeding of GAA mice. Four GAA heterozygous breeding pairs (F£ET; GAA ⁺ ; 6 ^neo /6 +) were obtained from Jackson Laboratories (Maine, USA) to generate homozygous GAA knock-outs (MUT; GAA ; 6 ^neo /6 ^neo ). Mice were genotyped at 1 month of age using a standard genotyping kit as per vendor's instructions (REDExtract-N-Amp Tissue PCR Kit; Sigma Aldrich). During breeding and throughout the experimental period, animals were housed in standard cages with 12-h light/dark cycles and free access to water/rodent chow (Harlan Teklad

8640 22/5) at McMaster University's Central Animal Facility. The study was approved by McMaster University's Animal Research and Ethics Board, and the experimental procedures strictly followed guidelines published by the Canadian Council of Animal Care.

[001 15] Production of Non-immunugenic GAA protein-loaded Exosomes for Treatment of GAA mice. Mouse GAA protein was produced as described previously in Example 1. To produce exosomes from mouse dendritic cells (DC), DC were harvested also as described in Example 1. GAA protein was loaded into the exosomes (unmodified) using the protein loading method as in Example 1. GAA protein-loaded exosomes were resuspended in 0.9% saline solution for treatment. GAA mice were divided into three treatment groups (n = 7 per group): saline, naked GAA protein, and GAA protein-loaded exosome (EXO GAA). Mice were treated once a week for 7 weeks. 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.

[001 17] Extraction of total glycogen. Total glycogen content in tissues (tibialis anterior, heart, and brain) was determined as previously described (Devries et al., 2005, Adamo et al., 1998). Briefly, snap-frozen tissue samples were freeze dried for 48 h, powdered, and dissected free of any blood and connective tissue and weighed. Ice-cooled 1 M HCl (100 iL) was added to 2-5 mg of tissue powder in 5 mL glass tubes, followed by vortexing and pressing the tissue powder against the tube walls with a glass rod. Prior to hydrolysis, tubes were weighed and sealed with glass stoppers, and thereafter placed in a water bath (100 °C) for 2 h, after which the tubes were reweighed and any change in weight was rectified with the addition of deionized water. Lastly, hydrolyzed samples were neutralized with 2 M Trizma base, vortexed, centrifuged at 3000 rpm for 5 min, transferred to 1.5 mL polyethylene tubes, and stored at - 80 °C. [00118] Fluorometric analysis of total glycogen. Total glycogen content in the tissue samples (tibialis anterior, heart, and brain) 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 μΜ-600 μΜ) and neutralized samples was loaded in triplicate onto a 96 well black plate. Next, 190 of assay buffer (50 mM Tris, 1 mM MgCl ₂ , 0.5 mM DTT, 300 μΜ ATP, 50 μΜ NADP, 0.02 U/mL glucose-6-P dehydrogenase in DW, pH 8.1) was added to each well, which was followed by a 5-min incubation step in the dark and a baseline reading at 340 nm excitation/460 nm emission. Thereafter, the final reaction was commenced by adding 5 iL hexokinase to each sample and the plate was incubated for 30 min at RT as described previously. 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.

Results

[00119] 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)). Additionally, 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). [00120] GAA protein-loaded exosome therapy restores GAA activity in all tissue tested vs. conventional naked GAA ERT in GAA KO mice. 7-week treatment in GAA KO mice with GAA protein-loaded exosomes restored GAA activity in slow-, fast-, and mixed fiber type skeletal muscle, diaphragm, and heart to a greater degree (Figure 10B) than conventional naked GAA ERT (Figure 10A). Importantly, GAA activity was restored in brain confirming that GAA protein-loaded exosomes cross the blood-brain barrier and restores GAA enzyme activity in neuronal tissue (brain, Figure 10B).

[00121] 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.

Example 3 - Treatment of GAA deficiency in vivo with mRNA-loaded Exosomes

GAA mRNA-loaded exosomes therapy restores GAA activity and GAA mRNA in all tissue tested to wild-type mice levels

[00122] A 7-week intravenous treatment of GAA knockout (KO) mice (as above) with

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.

[00123] Treatment of GAA KO mice with GAA mRNA-loaded exosomes restored GAA activity in fast- (EDL) and slow- (soleus) fiber-type skeletal muscle, diaphragm, and heart, as compared to that in GAA KO mice treated with empty exosomes (Figures 12A and 12B). Empty exosomes and GAA mRNA-loaded exosomes were given to wild-type mice (WT) as controls. GAA activity was restored in GAA KO brain depicting that GAA mRNA-loaded exosomes could not only cross the blood-brain barrier, but GAA mRNA delivered via exosomes did induce functional changes in neuronal tissue (Figure 13). It should be noted that the 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). It was concluded that the effect of the mRNA (packaged in exosomes) therapy persists for much longer periods of time as compared to conventional enzyme replacement therapy. Additionally, GAA mRNA-loaded exosomes resulted in substrate reduction (total glycogen content) in skeletal muscle (quadriceps and tibialis anterior) and heart in GAA KO mice to normal physiological wild-type levels (Figure 15).

[00124] Interestingly, 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

[00125] The 7-week treatment of GAA mRNA-loaded exosomes in GAA KO mice normalized grip endurance in the wire hang test (marker of neuromuscular function) to wild-type mice levels, while grip endurance in GAA KO mice treated with empty exosomes was not restored (control group; Figure 16). Wild-type mice treated with GAA mRNA-loaded exosomes maintained normal grip endurance (Figure 16). Example 4 - Treatment of Niemann-Pick disease, type CI deficiency

[00126] To determine the utility of the present exosomes to treat lysosomal storage disease that primarily affects the CNS (i.e., Niemann-Pick type C, neural ceroid lipofuscinosis, Krabbe disease, metachromatic leukodystrophy, etc.), the following studies were conducted.

[00127] An in vitro study was conducted in which primary dermal fibroblasts from three patients with genetically confirmed Niemann-Pick disease, type CI (NPCl) mutations were treated for 48 h as follows: media control (no therapy), "empty" exosomes, exosomes + human rNPCl mRNA (NPCl mRNA was cloned from the human genome (NCBI Accession #: NM_000271.4), and exosomes + human rNPCl protein (human rNPCl mRNA was cloned from human genome and expressed in CHO cells). 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.

[00128] Outcome measured was filipin staining (showing the accumulation of cholesterol and glycosphingolipids). A complete restoration of normal filipin staining in patient cells treated with NPCl mRNA-loaded exosomes, and near complete restoration of filipin staining in patient cells treated with NPCl protein-loaded exosomes resulted indicating NPCl restoration (Figure 17).

Example 5 - Treatment of Niemann-Pick disease, type CI in vivo

[00129] Breeding of NPCl mice. Four NPCl heterozygous breeding pairs (HET; NPC ⁺ ; 6 ^ne 76 +) were obtained from Jackson Laboratories (Maine, USA) to generate homozygous NPCl knock-outs (MUT; NPCT^; 6 ^neo /6 ^neo ). Mice were genotyped at 1 month of age using a standard genotyping kit as per vendor's instructions (REDExtract-N-Amp Tissue PCR Kit; Sigma Aldrich). During breeding and throughout the experimental period, animals were housed in standard cages with 12-h light/dark cycles and free access to water/rodent chow (Harlan Teklad 8640 22/5) at McMaster University's Central Animal Facility. The study was approved by McMaster University's Animal Research and Ethics Board, and the experimental procedures strictly followed guidelines published by the Canadian Council of Animal Care. [00130] 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.

[00131] At 30 days of age, NPCl homozygous mice and littermate wildtype mice

(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.

[00132] The 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.

Example 6 - Treatment of β-glucocerebrosidase deficiency

[00133] 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, β-glucocerebrosidase or glucosylceramidase (GBA). The following experiment is conducted to determine if this enzyme could be delivered to cells using exosomes.

[00134] Primary dermal fibroblasts grown from skin biopsies obtained from three Gaucher patients and three healthy age/gender-matched controls are treated with 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. Treatment is with 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.

[00135] 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.

[00136] Remarkably, 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. Thus, 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.

[00137] Given the demonstration that 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.

[00138] Relevant portions of references referred to herein are incorporated by reference.