WO2017147719A1 - Method for treating neuropathy - Google Patents

Abstract

A method of treating a neuropathy, such as a recessive neuronopathy or peripheral neuropathy, in a mammal is provided. The method comprises administering to the mammal a therapeutically effective amount of exosomes genetically engineered to comprise a functional neuropeptide or nucleic acid encoding the neuropeptide.

Description

METHOD FOR TREATING NEUROPATHY

Field of the Invention

[0001] The present invention generally relates to treatment of a neuropathy, including for example, an autosomal- or X-linked- recessive neuronopathy or peripheral neuropathy, using exosomes.

Background of the Invention

[0002] Hereditary neuronopathies and peripheral neuropathies encompass a large group of disorders, which share a common origin of pathology based on a deficiency or dysfunction of a protein due to mutations in one or more genes encoding a protein found in a motor or sensory peripheral nerve. Symptoms of hereditaiy neuronopathies and peripheral neuropathies vary considerably between the individual disorders and disease severity, but generally involve conditions relating to impaired motor function (e.g. muscle atrophy and limb weakness, respiratory distress), impaired sensory function (e.g. loss of sensation in hands and feet) and impaired autonomic function (e.g. dysregulated blood pressure and temperature regulation). These groups of disorders include genetic mutations that are either dominant or recessive in nature. Unlike autosomal dominant disorders that often have a dominant negative effect, an important consideration in both autosomal recessive and X-linked recessive disorders is that genetic replacement of the mutant allele would constitute a functional cure by restoring the normal function of the gene. Given that carriers of autosomal recessive disorders do not have clinical or functional consequences, a recovery of at least 50% of the relevant protein activity would constitute a cure. Similarly, female carriers of X-linked recessive disorders without skewed X- inactivation (> 90 %) do not have clinical or functional consequences, implying that the restoration of > 10 % of the relevant protein would constitute a cure.

[0003] One of the most common types of autosomal recessive disorders is the neuronopathy, spinal muscular atrophy (SMA), which is caused by mutations in the survival of motor neuron 1 , telomeric (SMNl) gene and the subsequent deficiency of full length survival of motor neuron (SMN) protein, In the absence of the SMN protein, SMA patients experience severe muscle wasting and loss of mobility, In the most severe form (SMA0), children are born with muscle weakness that is so severe they die in infancy; whilst, SMA1 children usually die by 3 years of age, SMA2 children never walk and can survive with severe disability into adulthood, SMA3 patients can walk and then lose the ability later in life, and SMA4 patients have the onset of muscle weakness in adulthood. Other recessive neuronopathies that are caused by a single gene mutation include hereditary amyotrophic lateral sclerosis (ALS) and hereditary spastic paraparesis (HSP). Two examples of recessive peripheral neuropathies include the groups of hereditary motor and sensory neuropathies (HMSN), which are also known as Charcot-Marie-Tooth disorders, and hereditary sensory and autonomic neuropathies (HSAN). HMSN disorders can be caused my mutations in many different genes (such as PMP22 and MFN2), leading to defects in the myelination of neurons or the axonal structure/function and comprise one of the most common hereditary neuromuscular disorders.

[0004] Owing to the inherent difficulty in correcting recessive neuronopathies and peripheral neuropathies resulting from gene mutations, there are presently no cures available. The current mainstay of treatment is thus based on supportive care designed to treat individual symptoms of the disorders and enhance the quality of life for patients. Examples of present treatments include correctional surgery, the use of orthopaedic devices, respiratory therapy, extensive medical counselling and pain medications.

[0005] In addition to these supportive treatments, numerous gene therapy-based methods have been investigated in an attempt to cure recessive neuronopathies and peripheral neuropathies, but these approaches have not adequately fulfilled the requirements of a desirable therapy due to fundamental issues such as: high immunogenicity, toxicity, the induction of an inflammatory response, the promotion of tumorigenesis and low to nil therapeutic efficacy, among others.

[0006] Thus, there is a need to develop improved methods of treating neuropathies such as recessive neuronopathies and peripheral neuropathies.

Summary of the Invention

[0007] It has now been determined that exosomes may be effectively used as a vehicle to deliver nucleic acid encoding a protein to a mammal to treat pathological neuropathies such as recessive neuronopathies and peripheral neuropathies that result from a deficiency of a functional protein. [0008] Thus, in one aspect of the invention, a method of treating a neuropathy is provided comprising administering to the mammal exosomes that are genetically modified to incorporate a nucleic acid encoding a functional neuropeptide.

[0009] In another aspect, a method of increasing the amount of a functional neuropeptide in a mammal is provided, comprising administering to the mammal exosomes that are genetically modified to incorporate a nucleic acid encoding the neuropeptide and/or the neuropeptide.

[0010] In another aspect, a method of increasing the activity of a target neuropeptide in a mammal is provided, comprising administering to the mammal a composition comprising exosomes which are genetically modified to incorporate nucleic acid encoding the functional neuropeptide and/or the neuropeptide.

[0011] In a further aspect, exosomes genetically engineered to incorporate nucleic acid encoding a neuropeptide and/or a neuropeptide are provided. Additional aspects of the invention include aspects and variations set forth in the following lettered paragraphs:

[0012] Al . An exosome produced by a process that comprises^*, (a) isolating exosomes from a biological sample from an organism (autologous) or from a conditioned medium from a cultured cell (allogenic or xenogenic); and (b) introducing a modification into the exosome selected from the group consisting of:

(i) at least one nucleic acid comprising a nucleotide sequence that encodes a functional neuropeptide or precursor thereof;

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

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

(iv) two or more of (i), (ii) and (iii). [0013] A2. The exosome according to paragraph Al, wherein the isolating includes a step of precipitating exosomes with polyethylene glycol, and resuspending the exosomes in a saccharide solution such as a trehalose solution.

[0014] A2.1 The exosome according to any one of paragraphs Al or A2, wherein the isolating removes vesicles that are greater than 140 nm in diameter.

[0015] 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.

[0016] 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.

[0017] A5. An exosome that comprises a modification selected from the group consisting of:

(i) at least one nucleic acid comprising a nucleotide sequence that encodes a functional neuropeptide or precursor thereof;

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

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

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

[0018] Bl . The exosome according to any of paragraphs Al - A5, having a diameter of 20-140 nm.

[0019] B2. The exosome according to any of paragraphs Al - A5, that comprises a nucleic acid comprising a nucleotide sequence encoding a functional neuropeptide or precursor thereof, wherein the nucleic acid is present in a lumen of the exosome. [0020] B2.1. The exosome according to paragraph B2, wherein the nucleic acid comprises a species of RNA or a species of modified RNA (modRNA, e.g. 5 methyl cytosine, or N6 methyladenine) encoding for a protein set forth in Table 1 and/or Table 2.

[0021] B3. The exosome according to paragraph B2 or B2.1, wherein the protein comprises one or more of the proteins set forth in Table 1 and/or Table 2.

[0022] B4, The exosome according to paragraph B3, wherein the protein is an enzyme.

[0023] B5. The exosome according to any one of paragraphs B2 - B2.1, wherein the nucleic acid encoding for the protein is selected from the group consisting of survival of motor neuron 1, telomeric (SMN1), vaccinia related kinase 1 (VRK1), exosome component 3 (EXOSC3), exosome component 8 (EXOSC8), immunoglobulin mu binding protein 2 (IGHMBP2), DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2), pleckstrin homology and RhoGEF domain containing G5 (PLEKHG5), ubiquitin like modifier activating enzyme 1 (UBAl), ATPase copper transporting alpha (ATP7A), LAS 1 -like, ribosome biogenesis factor (LASIL), heat shock protein family B (small) member 1 (HSPB 1), histidine triad nucleotide binding protein 1 (HINT1), ALS2, alsin Rho guanine nucleotide exchange factor (ALS2) , spastic paraplegia 1 1 (autosomal recessive) (SPG11), optineurin (OPTN), sigma non-opioid intracellular receptor 1 (SIGMAR1), solute carrier family 52 (riboflavin transporter), member 3 (SLC52A3) , chromosome 12 open reading frame 65 (cl2orf65), spastic paraplegia 7 (pure and complicated autosomal recessive) (SPG7) , ER lipid raft associated 2 (ERLIN2), proteolipid protein 1 (PLP1), LI cell adhesion molecule (LICAM), solute carrier family 16, member 2 (thyroid hormone transporter) (SLC16A2), adenosine monophosphate deaminase 2 (AMPD2), adaptor related protein complex 5 zeta 1 subunit (AP5Z1), ADP ribosylation factor like GTPase 6 interacting protein 1 (ARL6IP1), arylsulfatase family member I (ARSI), chromosome 10 open reading frame 2 (C10orf2), chaperonin containing TCP1 subunit 5 (CCT5), cytochrome P450 family 2 subfamily U member 1 (CYP2U1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), DDHD domain containing 1 (DDHD1), DDHD domain containing 2 (DDHD2), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), ER lipid raft associated 1 (ERLINl), fatty acid 2-hydroxylase (FA2H), fibronectin leucine rich transmembrane protein 1 (FLRT1), gap junction protein gamma 2 (GJC2), kinesin family member 1A ( IF1A), kinesin family member 1C (KIF1C), methionyl-tRNA synthetase (MARS), 5'- nucleotidase, cytosolic II (NT5C2), post-GPI attachment to proteins 1 (PGAP1), phospholipase A2 group VI (PLA2G6), patatin like phospholipase domain containing 6 (PNPLA6), polymerase (DNA directed), gamma (POLG), RAB3 GTPase activating non-catalytic protein subunit 2 (RAB3GAP2), sacsin molecular chaperone (SACS), ganglioside induced differentiation associated protein 1 (GDAP1), myotubularin related protein 2 (MTMR2), SET binding factor 1 (SBF1), SET binding factor 2 (SBF2), SH3 domain and tetratricopeptide repeats 2 (SH3TC2), N- myc downstream regulated 1 (NDRG1), early growth response 2 (EGR2), periaxin (PRX), hexokinase 1 (H 1), FYVE, RlioGEF and PH domain containing 4 (FGD4), FIG4 phosphoinositide 5-phosphatase (FIG4), surfeit 1 (SURF1), peripheral myelin protein 22 (PMP22), N-acylsphingosine amidohydrolase (acid ceramidase) 1 (ASAH1), phosphomannomutase 2 (PMM2), peroxisomal biogenesis factor 1 (PEX1), peroxisomal biogenesis factor 7 (PEX7), abhydrolase domain containing 12 (ABHD12), DnaJ heat shock protein family (Hsp40) member C3 (DNAJC3), phytanoyl-CoA 2-hydroxylase (PHYH), amin A/C (LMNA), mediator complex subunit 25 (MED25), leucine rich repeat and sterile alpha motif containing 1 (LRSAM1), tripartite motif containing 2 (TRIM2), polynucleotide kinase 3'- phosphatase (PNKP), KIF1A, solute carrier family 12 (potassium/chloride transporter), member 6 (SLC12A6), SCYl-like, kinase-like 1 (SCYL1), tyrosyl-DNA phosphodiesterase 1 (TDP1), PLA2G6, mitofusin 2 (MFN2), receptor accessory protein 1 (REEPl), neurofilament, light polypeptide (NEFL), gigaxonin (GAN), solute carrier family 25 member 46 (SLC25A46), gap junction protein beta 1 (GJB1), pyruvate dehydrogenase kinase 3 (PDK3), apoptosis inducing factor, mitochondria associated 1 (AIFM1), phosphoribosyl pyrophosphate synthetase ί (PRPS1), WNK lysine deficient protein kinase 1 (WIN 1), family with sequence similarity 134 member B (FAM134B), sodium voltage-gated channel alpha subunit 9 (SCN A), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein (IKBKAP), neurotrophic tyrosine kinase, receptor, type 1 ( TRK1), nerve growth factor (beta polypeptide) (NGF), dystonin (DST), PR domain 12 (PRDM12), clathrin heavy chain like 1 (CLTCL1), feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), and tectonin beta-propeller repeat containing 2 (TECPR2). [0024] B6. The exosome according to any one of paragraphs B2 - B5_}- further comprising at least one fusion product comprising a nerve targeting sequence linked to an exosomal membrane marker.

[0025] B7. The exosome according to any one of paragraphs Al - A5 or Bl, that comprises at least one fusion product comprising a nerve targeting sequence linked to an exosomal membrane marker.

[0026] B8. The exosome according to paragraph B6 or B7, wherein the exosomal membrane marker is selected from the group consisting of CD9, CD37, CD53, CD63, CD81 , CD82, CD151, an integ in, 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.

[0027] B9. The exosome according to any one of paragraphs B6 - B8, wherein the nerve targeting sequence is selected from the group consisting of myelin-associated glycoprotein, kinesin-like protein 1A, synthaxinl, synaptosomal-associated protein 25kDa, synaptobrevin and fragments thereof.

[0028] B10. The exosome according to any one of paragraphs B6 - B9, wherein the fusion product is a fusion protein.

[0029] Bl l. The exosome according to paragraph B10, further wherein the fusion protein includes a peptide linker between the nerve targeting sequence and the exosomal membrane marker,

[0030] B12. The exosome according to any one of paragraphs B6-B10, wherein the fusion product includes a transmembrane domain and localizes in a membrane of the exosome.

[0031] CI . A composition comprising exosomes according to any one of paragraphs

Al— A5, and a pharmaceutically acceptable carrier. [0032] C2. The composition according to paragraph CI, wherein the composition is substantially free of vesicles having a diameter less than 20 nm.

[0033] C3. The composition according to paragraph CI or C2, wherein the composition is substantially free of vesicles having a diameter greater than 140 nm.

[0034] Dl . A method of increasing the amount of a neuropeptide in a mammal, comprising administering to the mammal an exosome according to any one of paragraphs Al - B12, or a composition according to any one of paragraphs CI - C3.

[0035] D2. Use of an exosome according to any one of paragraphs Al - B12, or a composition according to any one of paragraphs CI - C3, for increasing the amount of a neuropeptide in a mammal.

[0036] D3. A method of treating a neuropathy in a mammal comprising administering to the mammal an exosome according to any one of paragraphs Al - B12, or a composition according to any one of paragraphs CI - C3.

[0037] D4. Use of an exosome according to any one of paragraphs Al - B12, or a composition according to any one of paragraphs CI - C3, for treating recessive neuronopathies and peripheral neuropathies in a mammal.

[0038] D5. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human.

[0039] D6. The method or use according to paragraph D5, wherein the human has a recessive neuronopathy or peripheral neuropathy selected from the group consisting of spinal muscular atrophy (SMA) type 0, SMA type 1, SMA type 2, SMA type 3, SMA type 4, SMA with pontocerebellar hypoplasia (PCH), distal SMA, Distal Hereditary Neuropathy (HMN), amyotrophic lateral sclerosis (ALS), hereditary spastic paraparesis (HSP), hereditary motor and sensory neuropathies (HMSN) and hereditary sensory and autonomic neuropathy (HSAN).

[0040] 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/or Table 2, and the exosome comprisesa nucleic acid encoding a neuropeptide as set in Table 1 and/or Table 2 corresponding with the disease.

[0041] These and other aspects of the invention will be described by reference to the following figures.

Brief Description of the Figures

[0039] Figure 1 graphically illustrates expression levels of lucifei ase delivered in vivo to the sciatic nerve via lucifeiase mRNA-loaded exosomes.

Detailed Description of the Invention

[0042] A method of treating a neuropathy such as recessive neuronopathies and peripheral neuropathies in a mammal is provided in which the neuropathy results from a nucleic acid mutation that results in a dysfunctional protein or lack of a protein. The method comprises administering to the mammal a therapeutically effective amount of exosomes engineered to incorporate nucleic acid encoding a functional target neuropeptide or the target neuropeptide.

[0043] The term "neuropathy" is used broadly herein to refer to any disease that affects the peripheral nervous system. This includes disorders that affect the cell body of neurons (such as myelinopathies and axonopathies), and neuronopathies that result from neuron degeneration, including sensory neuronopathy, motor neuronopathy and autonomic neuronopathy.

[0044] The term "neuropeptide" is used herein to refer to proteins or peptides which function to modulate neural cell activity in the nervous system. Neuropeptides may be produced by neurons, and may include neurotransmitters, mitochondrial proteins, cytoskeletal proteins, cytosolic proteins, peroxisomal proteins, lysosomal proteins, chemokines, growth factors and peptide hormones

[0045] The term "functional" with respect to a target neuropeptide is used herein to refer to a protein product which retains innate biological activity, including but not limited to, catalytic, metabolic, regulatory, binding, transport and the like. As will be appreciated by one of skill in the art, to be functional, a target protein need not exhibit an endogenous level of biological activity, but will exhibit sufficient activity to render it useful to treat a neuropathy such as a recessive neuronopathy and peripheral neuropathy, e.g, at least about 10% of the biological activity of the corresponding endogenous protein, and preferably at least about 25-50% or greater of the biological activity of the corresponding endogenous protein. For autosomal recessive disorders, it is preferred that a functional protein possess at least about 25% of the biological activity of the corresponding endogenous protein, and more preferably at least about 50% or greater. For X- linked recessive disorders, it is preferred that a functional protein possess at least about 10% of the biological activity of the corresponding endogenous protein, and preferably at least about 25% or greater of the biological activity of the corresponding endogenous protein.

[0046] The term "exosome" refers to cell-derived vesicles having a diameter of between about 20 and 140 nm, for example, a diameter of about 40-120 nm, including exosomes with a mean diameter of about 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 and/or 120 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, tumor 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 that distinguish them from 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 integ ins, ICAM-1, EpCAM and CD31 ; membrane fusion markers such as annex ins, 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.

[0047] Exosomes may be obtained from an appropriate biological sample using a combination of isolation techniques, for example, centrifugation, filtration and ultracentdfugation methodologies, as well as PEG-based methods. In one embodiment, exosomes may be isolated from a biological sample using a method including the steps of: i) optionally 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) optionally subjecting the supernatant from step i) to centrifugation to remove micro vesicles and apoptotic bodies therefrom; iii) optionally microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) combining the microfiltered supernatant from step iii) with a polyethylene glycol solution to precipitate the exosomes and subjecting the solution to at least one round of centrifugation to obtain an exosome pellet; and v) re-suspending the exosome pellet from step iv) in a trehalose solution and conducting an optional centrifugation step to remove vesicles having a diameter of greater than 140 nm from the solution.

[0048] In accordance with an aspect of the present invention, the process of isolating exosomes from a biological sample includes a first optional 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 first 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 an additional 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. [0049] 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 12,000-15,000x 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.

[0050] The resulting supernatant is collected and subjected to a third optional 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.

[0051 ] The supernatant is then optionally 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.

[0052] The microfiltered supernatant (filtrate) may then be combined with a polyethylene glycol (PEG) solution to precipitate exosomes within the filtrate. As would be appreciated by one of skill in the art, a variety of PEG formulations may be used. Preferably, these formulations comprise PEG chain lengths having an average molecular weight of between about 400 to 20,000 daltons (e.g. 1000 to 10,000 daltons, such as 6000 daltons). Similarly, the exosome-PEG solutions may have varying final concentrations of PEG, for example, a final concentration of PEG may be between about 5-15% (such as 8%). Preferably, the filtrate is combined with an equal volume of the PEG solution, having a strength in the range of about 10-20% PEG. Salts may be added to the PEG solution to enhance the precipitation of exosomes. Preferably, a salt such as NaCl is added to the PEG solution so that the final concentration of salt in the exosome-PEG-salt solution is between about 50 to 1,000 mM (such as 500 mM). The PEG-filtrate is gently mixed and incubated under conditions suitable for exosome precipitation, e.g. incubated for 30 minutes at 4°C. Some samples may require a longer incubation period for exosome precipitation to occur,

[0053] Following incubation, the precipitated exosomes were pelleted by centrifugation, e.g. at 10,000x g for 10 min at 4°C, and the pellet was solubilized in a suitable saccharide solution, such as a trehalose solution, that is effective to reduce aggregation of the exosomes. The saccharide is preferably solubilized in a physiological buffer, such as saline or PBS. In one embodiment, a trehalose solution of various concentrations is effective at reducing the aggregation of exosomes, such as a trehalose concentration between 10 mM to 1,000 mM (e.g. 500 mM).

[0054] To remove non-exosome extracellular vesicles (i.e. vesicles larger than 140 nm), the trehalose exosome solution may be subjected to further optional centrifugation or ultracentrifugation steps, for example, at 15,000x g - 150,000x g for 1 hr at 4°C. If ultracentrifugation is performed, exosomes will be present in both the resultant pellet and supernatant fractions, generally with a larger quantity of exosomes in the supernatant.

[0055] To enhance removal of impurities that are smaller than the exosomes, e.g. smaller than 20nm, the exosome-trehalose solution may be subjected to an optional ultrafiltration step using either a direct-flow filtration technique (such as a centrifugal spin filter) or a cross-flow filtration technique (such as a tangential flow system). As would be appreciated by one of skill in the art, filtration membranes suitable for this step may possess a molecular weight cut-off (MWCO) rating in the range of 3-500kDa and preferably between 100-300kDa.

[0056] In another embodiment, exosome isolation may include 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 and apoptotic bodies therefrom; iii) microfiltermg 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.

[0057] The centrifugation and filtration steps (steps i)-iii)) are as previously described.

[0058] Following the initial centrifugation and filtration steps, 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.

[0059] 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 J 00- 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,

[0060] 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 appropriate sucrose fractions are collected and may be combined with other collected sucrose fractions, and the resuspended exosome pellet is ready for use, or may preferably be subjected to an uitracentrifugation 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 (Percoll™) or a iodixanol density gradient, then the resuspended exosome pellet may be subjected to additional wash steps, e.g. subjected to one to three uitracentrifugation 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. The pellet is removed from the supernatant and may be re-suspended in a physiologically acceptable solution for use.

[0061] As one of skill in the art will appreciate, the exosome pellet from any of the centrifugation or uitracentrifugation steps may be washed between centrifugation steps using an appropriate physiological solution, e.g. sterile PBS, sterile 0.9% saline or sterile carbohydrate- containing 0.9% saline buffer.

[0062] The present methods advantageously provide a means to obtain mammalian and non-mammalian exosomes which are useful therapeutically. In some embodiments, the methods yield exosomes which exhibit a high degree of purity, for example, at least about 50% pure, and preferably, at least about 60%, 70%, 80%, 90% or 95% or greater pure. Preferably, the exosomes are "essentially free" from cellular debris, apoptotic bodies and microvesicles having a diameter less than 20 nm or greater than 140 nm, and preferably less than 40 nm 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 degree of stability, that may be evidenced by the zeta potential of a mixture/solution of such exosomes, for example, a zeta potential of at least a magnitude of ±10 mV, e.g. < -10 or > +10, and preferably, a magnitude of at least 20 mV, 30 mV, 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, generally the higher the magnitude of the zeta potential, the greater the stability of the exosomes. [0063] Moreover, high quantities of exo somes are achievable by the present isolation method. With the PEG-based method, lmL of serum yields about 5-10 mg of protein. With the ultracentrifugation/density gradient method, 1 mL of serum or 15-20 mL of cell culture spent media (from at least about 2 x 10⁶ cells) yields about 100-2000 μg total protein. Thus, solutions comprising exosomes at a concentration of at least about 5 μg μL, and preferably at least about 10-25 pg^L, may readily be prepared due to the high exosome yields obtained by the present method. The term "about" as used herein with respect to any given value refers to a deviation from that value of up to 10%, either up to 10% greater, or up to 10% less.

[0064] Exosomes isolated in accordance with the methods herein described, beneficially retaining integrity, and exhibiting purity (being "essentially free" from entities having a diameter less than 20 nm and or greater than 140 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. They have also been determined to be non- allergenic, and thus, safe for autologous, allogenic, and xenogenic use.

[0065] For the treatment of neuropathies, such as recessive neuronopathies or peripheral neuropathies, isolated exosomes are genetically engineered to incorporate exogenous nucleic acid suitable to treat the disease, for example, nucleic acid (e.g. DNA, or mRNA) encoding a functional neuropeptide, or to incorporate the neuropeptide itself. The term "exogenous" is used herein to refer to a nucleic acid or protein originating from a source external to the exosomes. The desired nucleic acid may be produced using known synthetic techniques andincorporated 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 other transfection methods. More particularly, electroporation applying voltages in the range of about 20-1000 V/cm may be used to introduce nucleic acid 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 μΐ, to 10 may be used to load 100 ng to 2500 ng nucleic acid or protein into exosomes. Other methods may also be used to load nucleic acid or protein into exosomes including, for example, the use of cell-penetrating peptides.

[0066] 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 nucleic acid in an amount of at least about 1 ng nucleic acid (e.g. mRNA) per 10 ug of exosomal protein, or at least about 30 ug protein per 10 ug of exosomal protein.

[0067] In another embodiment, a nucleic acid-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, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like, by electrop oration or other transfection method as described above. Following a sufficient period of time, e.g. 3-7 days to achieve stable expression of the nucleic acid, exosomes incorporating the expressed nucleic acid may be isolated from the exosome-producing cells as described herein.

[0068] The desired nucleic acid encoding a neuropeptide, or the neuropeptide, may be introduced into isolated exosomes, as previously described, using electroporation or other transfection methods. Introduction to the exosome of both the desired neuropeptide and nucleic acid encoding the same neuropeptide may increase delivery efficiency of the neuropeptide. In addition, introduction of a combination of neuropepeptides and/or nucleic acids encoding one or more neuropeptides may be desirable to treat a recessive neuronopathy or peripheral neuropathy resulting from different DNA mutations, or for the treatment of secondary pathologies such as mitochondrial dysfunction in neuropathy associated with type 2 diabetes.

[0069] In another embodiment, prior to incorporation into exosomes nucleic acid encoding a selected protein, or incorporation of the protein, exosomes may be modified to express or incorporate a target-specific fusion product which provides targeted delivery of the exosomes to nerve cells. Such a target-specific fusion product comprises a sequence that targets nerves, i.e. a nerve 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 LIMP (lysosomal integral membrane protein). All or a fragment of an exosomal membrane marker may be utilized in the fusion product, provided that the 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 membrane marker into the exosomal membrane.

[0070] The target-specific fusion product also includes a nerve targeting sequence, i.e. a protein or peptide sequence which facilitates the targeted delivery of the exosome to nerves. Examples of suitable nerve targeting proteins include, but are not limited to, myelin-associated glycoprotein, kinesin-like protein 1A, synthaxinl, synaptosomal-associated protein 25kDa and synaptobrevin, or a targeting fragment thereof, e.g. a portion of the C-terminal sequence thereof.

[0071] Exosomes incorporating a nerve targeting fusion product may be produced, as described above, using recombinant technology. In this regard, an expression vector encoding the fusion product is introduced by electroporation or other transfection methods 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. The desired nucleic acid encoding the neuropeptide and/or the neuropeptide, may be introduced into isolated exosomes incorporating a nerve targeting fusion product (modified nerve targeting exosomes) as previously described, using electro oration or other trans ection methods. Exosomes incorporating the nerve targeting sequence and the desired nucleic acid encoding the neuropeptide and/or the neuropeptide may exhibit increased delivery efficiency of the neuropeptide and/or nucleic acid encoding the neuropeptide.

[0072] Exosomes genetically engineered to incorporate nucleic acid encoding a neuropeptide and/or the neuropeptide, may be used to deliver the nucleic acid and/or neuropeptide to a mammal in vivo in the treatment of a neuropathy, to upregulate the activity of the target protein and thereby treat the disease. For example, the present method may be used to treat any form of recessive neuronopathy or peripheral neuropathy resulting from a recessive genetic mutation. The term "mutation" is used herein to describe any inherited or sporadic change in the nucleotide sequence or arrangement of DNA that results in a dysfunctional or absent neuropeptide including, but not limited to the following: nucleotide substitutions (e.g. missense mutations, nonsense mutations, RNA processing mutations, splice-site mutations, regulatory mutations, nucleotide transitions and nucleotide transversions), insertions or deletions of one or more nucleotides, duplications of any nucleotide sequence, repeat expansion mutations (e.g. trinucleotide repeats, etc) and frameshift mutations. The term "recessive" is used herein to describe any X-linked recessive mutation or disorder or autosomal recessive mutation or disorder.

[0073] Examples of recessive neuronopathies that are caused by genetic mutations and that may be treated using the present engineered exosomes are set out in Table 1 below. Table 1 identifies the disease and affected or mutated gene involved in each disease, the type of mutation, the mRNA transcript sequence information (via the NCBI (National Centre for Bioteciinology Information) GenBank accession numbers) for the functional gene (which could be incorporated into the exosomes to treat a disease), and the corresponding protein sequence information for the proteins useful to treat each disease.

Table 1.

Disease Type of Affected Gene GenBank GenBank

mutation Accession protein

Number of Accession

Treatment Number

mRNA

[0074] Examples of recessive peripheral neuropathies that are caused by genetic mutations and that may be treated using the present engineered exosomes are set out in Table 2 below. Table 2 identifies the disease and affected or mutated gene involved in each disease, the type of mutation, the mRNA transcript sequence information (via the NCBI (National Centre for Biotechnology Information) GenBank accession numbers) for the functional gene (which could be incorporated into the exosomes to treat a disease), and the corresponding protein sequence information for the proteins useful to treat each disease.

Table 2.

[0075] Thus, in one embodiment, exosomes are used to deliver to a mammal one or more nucleic acids selected from the group consisting of survival of motor neuron 1, telomeric (SMN1), vaccinia related kinase 1 (VRKl), exosome component 3 (EXOSC3), exosome component 8 (EXOSC8), immunoglobulin mu binding protein 2 (IGHMBP2), DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2), pleckstrin homology and RhoGEF domain containing G5 (PLEKHG5), ubiquitin like modifier activating enzyme 1 (UBAl), ATPase copper transporting alpha (ATP7A), LAS 1 -like, ribosome biogenesis factor (LAS1L), heat shock protein family B (small) member 1 (HSPBl), histidine triad nucleotide binding protein 1 (HI T1), ALS2, alsin Rho guanine nucleotide exchange factor (ALS2) , spastic paraplegia 11 (autosomal recessive) (SPG11), optineurin (OPT ), sigma non-opioid intracellular receptor 1 (SIGMAR1), solute carrier family 52 (riboflavin transporter), member 3 (SLC52A3) , chromosome 12 open reading frame 65 (cl2orf65), spastic paraplegia 7 (pure and complicated autosomal recessive) (SPG7) , ER lipid raft associated 2 (ERLIN2), proteolipid protein 1 (PLP1), LI cell adhesion molecule (LI CAM), solute carrier family 16, member 2 (thyroid hormone transporter) (SLC16A2), adenosine monophosphate deaminase 2 (AMPD2), adaptor related protein complex 5 zeta 1 subunit (AP5Z1), ADP ribosylation factor like GTPase 6 interacting protein 1 (ARL6IP1), arylsulfatase family member I (ARSI), chromosome 10 open reading frame 2 (C10orf2), chaperonin containing TCP1 subunit 5 (CCT5), cytochrome P450 family 2 subfamily U member 1 (CYP2U1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), DDHD domain containing 1 (DDHD1), DDHD domain containing 2 (DDHD2), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), ER lipid raft associated 1 (ERLINl), fatty acid 2-hydroxylase (FA2H), fibronectin leucine rich transmembrane protein 1 (FLRT1), gap junction protein gamma 2 (GJC2), kinesin family member 1A (KIF1A), kinesin family member 1C (KIF1C), methionyl-tRNA synthetase (MARS), 5'- nucleotidase, cytosolic II (NT5C2), post-GPI attachment to proteins 1 (PGAP1), phospholipase A2 group VI (PLA2G6), patatin like phospholipase domain containing 6 (PNPLA6), polymerase (DNA directed), gamma (POLG), RAB3 GTPase activating non-catalytic protein subunit 2 (RAB3GAP2), sacsin molecular chaperone (SACS), ganglioside induced differentiation associated protein 1 (GDAP1), myotubularin related protein 2 (MTMR2), SET binding factor 1 (SBF1), SET binding factor 2 (SBF2), SH3 domain and tetratricopeptide repeats 2 (SH3TC2), N- myc downstream regulated 1 (NDRG1), early growth response 2 (EGR2), periaxin (PRX), hexokinase 1 (HK1), FYVE, RlioGEF and PH domain containing 4 (FGD4), FIG4 phosphoinositide 5-phosphatase (FIG4), surfeit 1 (SURF1), peripheral myelin protein 22 (PMP22), N-acylsphingosine amidohydrolase (acid ceramidase) 1 (ASAH1), phosphomannomutase 2 (PMM2), peroxisomal biogenesis factor 1 (PEX1), peroxisomal biogenesis factor 7 (PEX7), abhydrolase domain containing 12 (ABHD12), DnaJ heat shock protein family (Hsp40) member C3 (DNAJC3), phytanoyl-CoA 2-hydroxylase (PHYH), amin A/C (LMNA), mediator complex subunit 25 (MED25), leucine rich repeat and sterile alpha motif containing 1 (LRSAM1), tripartite motif containing 2 (TRIM2), polynucleotide kinase 3'- phosphatase (PNKP), KIF1A, solute carrier family 12 (potassium/chloride transporter), member 6 (SLC12A6), SCYl-like, kinase-like 1 (SCYL1), tyrosyl-DNA phosphodiesterase 1 (TDP1), PLA2G6, mitofusin 2 (MFN2), receptor accessory protein 1 (REEP1), neurofilament, light polypeptide (NEFL), gigaxonin (GAN), solute earner family 25 member 46 (SLC25A46), gap junction protein beta 1 (GJBl), pyruvate dehydrogenase kinase 3 (PDK3), apoptosis inducing factor, mitochondria associated 1 (AIFM1), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), WNK lysine deficient protein kinase 1 (WINK1), family with sequence similarity 134 member B (FAM134B), sodium voltage-gated channel alpha subunit 9 (SCN9A), inhibitor of kappa light polypeptide gene enhancer in B -cells, kinase complex-associated protein (IKBKAP), neurotrophic tyrosine kinase, receptor, type 1 (NTRK1), nerve growth factor (beta polypeptide) (NGF), dystonin (DST), PR domain 12 (PRDM12), clathrin heavy chain like 1 (CLTCL1), feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), tectonin beta-propeller repeat containing 2 (TECPR2), or the protein encoded by one or more of these nucleic acids.

[0076] Accordingly, the present method is useful to treat neuropathies such as recessive neuronopathies and peripheral neuropathies selected from the group consisting of spinal muscular atrophy (SMA) type 0, SMA type 1, SMA type 2, SMA type 3, SMA type 4, SMA with pontocerebellar hypoplasia (PCH), distal SMA, Distal Hereditary Neuropathy (HMN), amyotrophic lateral sclerosis (ALS), hereditary spastic paraparesis (HSP), hereditary motor and sensory neuropathies (HMSN), hereditary sensory and autonomic neuropathy (HSAN) and peripheral neuropathies that occur in type 1 or 2 diabetes. As would be appreciated by one skilled in the art, new mutations that cause recessive neuronopathies and peripheral neuropathies are continually being discovered and thus, the present method may also be effective to treat recessive neuronopathy and peripheral neuropathy causing gene mutations which are yet to be identified as such.

[0077] As one of skill in the art will appreciate, the nucleic acid encoding a neuropeptide and/or the neuropeptide, for incorporation into exosomes according to the invention may be a functional native mammalian nucleic acid or protein, including for example, nucleic acid or protein from human and non-human mammals, or a functionally equivalent nucleic acid or neuropeptide. The term "functionally equivalent" refers to nucleic acid, e.g. mRNA, rRNA, tRNA, DNA, or cDNA, encoding a neuropeptide, and is meant to include any nucleic acid sequence which encodes a functional neuropeptide, including all transcript variants, variants that encode protein isoforms, variants due to degeneracy of the genetic code, artificially modified variants, and the like. Thus, 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. The term "functionally equivalent" is used herein also to refer to a protein which exhibits the same or similar function to the native protein (e.g. retains at least about 30% of the activity of 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. 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 a nerve targeting sequence or targeting fragments thereof, at the N-terminal end of the protein and the like. Suitable modifications will generally maintain at least about 70% sequence similarity with the active site and other conserved domains of the native neuropeptide, and preferably at least about 80%, 90%, 95% or greater sequence similarity.

[0078] Engineered exosomes incorporating nucleic acid encoding a neuropeptide, and/or the neuropeptide, 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 earner 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).

[0079] 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 formuiated 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). Oral compositions may additionally include adjuvants including sugars, such as lactose, trehalose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, colouring agents and flavouring agents may also be present. Exosome compositions for topical application may be prepared including appropriate earners. 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, antioxidants and other preservatives may be added to the composition to prevent microbial growth and/or degradation over prolonged storage periods.

[0080] The present engineered exosomes are useful in a method to treat a pathological neuropathy, e.g. a recessive neuronopathy or peripheral neuropathy. The terms "treat", "treating" or "treatment" are used herein to refer to methods that favourably alter recessive neuronopathies and peripheral neuropathies, including those that moderate, reverse, reduce the severity of, or protect against, the progression of recessive neuronopathies and peripheral neuropathies. Thus, for use to treat such a disease, a therapeutically effective amount of exosomes engineered to incorporate nucleic acid encoding the functional protein, useful to treat the disease, are administered to a mammal. The term "therapeutically effective amount" is an amount of exosome required to treat the disease, while not exceeding an amount that 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 the amount or activity of the target neuropeptide in the individual being treated by at least about 10%, and preferably an increase in activity of the target neuropeptide of greater than 10%, for example, at least 20%, 30%, 40%, 50% or greater. For example, in one embodiment, the dosage may be a dosage in an amount in the range of about 20 ng to about 200 mg of total exosomal protein for the delivery of RNA species such as mRNA, tRNA, rRNA, mi NA, SRP UNA, snRNA, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, or piRNA. In an exemplary embodiment, a dosage of exosomes sufficient to deliver about 1 ng kg to about 100 ug/kg of a nucleic acid (e.g. an RNA species), is administered to the mammal in the treatment of a target recessive neuronopathy or peripheral neuropathy. In another embodiment, the dosage may be a dosage of exosomes sufficient to deliver about 0.1 mg/kg to about 100 mg/kg of a neuropeptide is administered to the mammal in the treatment of a neuropathy. 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.

[0081] As will be appreciated by one of skill in the art, exosomes comprising nucleic acid encoding the protein, for example, to treat recessive neuronopathies and peripheral neuropathies, 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; anti-oxidants (i.e., coenzyme Q10, alpha lipoic acid, vitamin E, synthetic coenzyme Q10 analogues, resveratrol, N-acetylcysteine, etc), creatine monohyd ate, optimal glycemic control, and/or pain medications.

[0082] In another embodiment, the present method of treating neuropathy in a mammal may include administration to the mammal of exosomes (for example, isolated as described above), genetically modified to incorporate gene-silencing systems (e.g., siRNA) to reduce the expression of a mutated gene followed by administering to the mammal exosomes genetically modified to incorporate a protein useful to treat the neuropathy and/or nucleic acid encoding the protein.

[0083] In another aspect of the invention, a method of treating neuropathy in a mammal may include administering to the mammal exosomes genetically modified to incorporate genome- editing systems to correct the inherent primary mutation leading to the neuropathy. Genome editing may include gene insertions, deletions, modifications and gene silencing. Examples of nuclease genome editing systems include, but are not limited to, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease system, e.g. including a targeting gRNA and a CRISPR-associated (Cas) gene, such as CRISPR-Cas9, Transcription Activator-Like Effector Nucleases (TALEN) and mito-TALEN, ZFN Zinc-Finger Nucleases (ZFN) and aptamer-guided delivery of therapeutic nucleic acids, e.g. small interfering RNA, micro RNA, anti-microRNA, antagonist and small hairpin RNA.

[0084] In one embodiment, the exosome is genetically modified to express a CRISPR nuclease system, such as a CRISPR/Cas9 Type II genome editing system, including a Cas 9 nuclease, and a guide RNA (gRNA) comprising fusion of a targeting RNA sequence, crRNA (CRISPR RNA) and a trans-activating RNA (tracrR A), The crRNA and tracrRNA are related to the selected Cas nuclease such that the crRNA and tracrRNA are specific for and recognized by the selected Cas nuclease.

[0085] The targeting sequence of the guide RNA (gRNA) is a strand of RNA that is homologous to a region on a target gene, i.e. a gene to be edited or silenced, associated with a neuropathy. Target genes may be genes associated with genetic disease, including autosomal recessive and X-linked recessive neuropathies. The targeting RNA may comprise from 10-30 nucleotides, e.g. from 15-25 nucleotides, and may comprise a GC content of about 40-80%. The CRISPR system may be utilized to disrupt expression of a gene by insertion or deletion of nucleotides to disrupt the Open Reading Frame (ORF) of a target gene, or to introduce a premature stop codon therein. Non-Homologous End Joining (NHEJ) DNA repair may be used in this instance. The CRISPR system may also be used to edit (e.g. to correct a gene mutation) by homology directed repair in which the targeting RNA includes an editing region, e.g. a region that incorporates an edit to be incorporated into the target gene, flanked by a region of homology (homologous arms) on either side thereof. The size of the editing region is not particularly restricted, and may include a single nucleotide edit, or edits of up to 100 nucleotides or more. The targeting sequence of the gR A is selected such that it targets a site within the target gene that is proximal (e.g. within about 2-5 nucleotides or more) to a protospacer adjacent motif (PAM) located within the target gene. The PAM is recognized by the Cas nuclease and permits Cas nuclease binding. The homologous arms will generally increase in size with the size of the editing region, for example, for edits of less than about 50 nucleotides, the homologous arms may be in the range of about 100-150 nucleotides in length, while larger editing regions may incorporate homologous arms of about 200-800 nucleotides, or more. Edits may also be introduced using CRISPR which facilitate expression of a target gene, e.g. edits which introduce a transcription factor that promote gene expression. The gRNA additionally incorporates related crRNA and a tracrPvNA sequences, which interact and function to direct the Cas nuclease to the target gene and catalyze cleavage of the target gene by the Cas nuclease. As will be understood by one of skill in the art, while each of the crRNA, tracrRNA, and Cas nuclease sequences are related, these sequences may be native or mutated sequences, provided that any mutations thereof do not have an adverse impact on function. Methods for selection of suitable crRNA and tracrRNA sequences for use in gRNA are known in the art.

[0086] The Cas nuclease may, for example, be a Cas 9-based nuclease. Examples of a Cas

9 nuclease include wild-type Cas 9 (a double nickase) from Streptococcus pyogenes (SP), Staphylococcus aureus (SA), Neisseria meningitidis (NM), Streptococcus thermophilus (ST), and Treponema denticola (TD), as well as mutated recombinant Cas 9, e.g. mutated to function as a single nickase such as Cas9 D10A and Cas9 H840A, which may be used with 2 or more gRNAs to achieve a genome edit with increasing targeting efficiency that prevents non-specific genomic editing. Wild-type and single nickase Cas 9 may be used to edit genes, for example, that result in autosomal recessive or X-linked recessive neuropathies, in order to correct the mutation. The mutated Cas 9 may also be a nuclease-deficient Cas (for example, incorporating both D10A and H840A to inactivate nuclease function) which binds but does not cleave and thereby silences a gene. Nuclease-deficient Cas 9 may be used to treat an autosomal recessive neuropathy, to prevent or minimize expression of a dysfunctional mutated protein, which may interfere with the activity of the desired functional protein.

[0087] The targeting RNA is an RNA strand complementary to a site on the target gene which is 3-4 nucleotides upstream of a PAM sequence recognized by the Cas nuclease. The targeting RNA does not itself include a PAM sequence, PAM sequences differ for various Cas nucleases. For example, for Streptococcus pyogenes (SP), the PAM sequence is NGG; for S. aureus, the PAM sequence is NNGRRT or NNGRR(N); for Neisseria meningitides, the PAM sequence is NNNGATT; for Streptococcus thermophilus, the PAM sequence is N AGAAW; for Treponema denticola (TD), the PAM sequence is NAAAAC. "N" represents any nucleotide, W = weak (A or T) and R = A or G.

[0088] For introduction into exosomes, nucleic acid encoding a nuclease genome editing system, such as a selected CRISPR nuclease system including gRNA and a Cas nuclease, may be produced using known synthetic techniques and then incorporated into the same or different expression vectors under the control of an appropriate promoter. Suitable vectors for such expression are known in the art, Alternatively, expression vectors incorporating the selected genome editing system may be obtained commercially. Expression vectors incorporating the nuclease editing system may be introduced into exosomes using electroporation or transfection using cationic lipid-based transfection reagents. Alternatively, the components of the nuclease editing system may be introduced directly into exosomes as single-stranded (ss) DNA using similar introduction techniques, e.g. gRNA of CRISPR may be introduced into exosomes as ssDNA.

[0089] In another embodiment, Class 2 CRISPR technology (such as CRISPR-spCAS9-

HF) can be incorporated into exosomes and used as a gene editing system.

[0090] The use of engineered exosomes in a therapy to treat recessive neuronopathies and peripheral neuropathies advantageously results in delivery of nucleic acid (mRNA, rRNA and tRNA) and/or protein efficiently and safely to a neural cell to treat genetic defects. Thus, the use of exosomes overcomes the challenges of delivery of therapeutic agents to the peripheral nervous system. [0091] Embodiments of the invention are described in the following examples which are not to be construed as limiting.

Example 1 - Preparation of Exosomes for Use to Treat Recessive Neuronopathies and Peripheral Neuropathies

[0092] To determine the efficacy of the present engineered exosomes to treat autosomal recessive and X-linked recessive neuronopathies and peripheral neuropathies resulting from genetic mutations, exosomes were engineered to treat one of the most common and representative autosomal recessive diseases: spinal muscular atrophy (SMA).

[0093] Exosomes were isolated and loaded with mRNA encoding the survival of motor neuron (SMN) protein as follows.

[0094] Dendritic cells (DC) were isolated from mouse bone marrow progenitor cells and from human peripheral blood mononuclear ceils (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, ImM 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 IMDM 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 IMDB 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. [0095] 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 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 μηι filter, followed by filtration through a 0.22 μηι 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 μηι filter sterilized water) in an ultracentrifuge tube. This mixture was spun at 100,000x-170,000 gfor 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. Yield was about 1 x 10⁹ 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.

[0096] Purified exosomes were suspended in a 100-140 of pie-chilled electroporation buffer (1.5 mM potassium phosphate pH 7.2, 25 mM KC1, and 21% (vol/vol) OptiPrep) for SMN1 mRNA electroporation of exosomes. Introduction of SMNl mRNA into exosomes

[0097] Electroporation mixture is prepared by carefully mixing isolated exosomes and

SMNl mRNA in 1 :1 ratio in electroporation buffer. Electroporation is carried out in 0.4 mm electroporation cuvettes at 400 mV and 125 μΡ capacitance (pulse time 14 milliseconds (ms) for mRNA) using Gene Pulse XCell electroporation system (BioRad). After electroporation, exosomes are 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 exosonie administration, SMNl mRNA loaded exosomes are re-suspended in 5% (wt vol) glucose in 0.9% saline solution.

[0098] Alternatively, exosomes are loaded with SMNl mRNA using cationic lipid-based transfection reagents (Lipofectamine® MessengerMAX™ Transfection Reagent, Life Technologies). After transfection, exosomes are 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. Mouse and human SMNl mRNA and luciferase mRNA is purchased from Trilink Biotechnologies.

Example 2 - Exosomes Packaged with mRNA can be Transported into Tissues Affected by Spinal Muscular Atrophy

[0099] To confirm the capacity of exosomes isolated as described herein to load and deliver to the sciatic nerve, the loading and delivery of unmodified and modified RNA was conducted.

[00100] Exosomes (10 ug of total exosomal protein), obtained as described in Example 1, were loaded with 100 ng of luciferase mRNA. Loading was accomplished using a cation-based transfection reagent as described in Example 1. Luciferase mRNA loaded exosomes (10 ug of total exosomal protein suspended in sterile 0.9% saline) were intravenously administered to mice. Mouse sciatic nerve was then harvested immediately 4 hours following injection (3 mice per group). Luciferase activity was measured in tissue homogenates to quantify the amount of labeled mRNA in the sciatic nerve. Luciferase mRNA loaded exosomes demonstrated a significant increase in luciferase activity when compared to saline control mice (Figure 1). Thus, mRNA, e.g. mKNA-hiciferase was efficiently loaded into exosomes, delivered to nerve cells and translated into function protein, indicating that mRNA encoding for the SMN1 protein can be packaged into exosomes and used for the treatment of spinal muscular atrophy.

Example 3 - Treatment of SMN deficiency in vitro with mRNA-loaded Exosomes

[00101] Human primary dermal fibroblasts were isolated from skin biopsies of healthy subjects (referred to as Control/CON) and patients with spinal muscular atrophy (with homozygous SMN1 mutations) (n = 4 age/sex-matched per group). Fibroblasts are treated with SM 1 mRNA at a dose of about 100-150 ng of mRNA, 10 ug (total exosomal protein) of empty exosomes (exosome control), or exosomal SMNl mRNA in dose equivalent to delivery of 40 mg/kg SMNl, about 100-150 ng mRNA, in 10 ug of total exosomal protein) for 48 hours in pre- spun growth media devoid of bovine microvesicles and exosomes.

[00102] Immunohistochemistry using SMN antibody (Abeam) is carried out to measure SMN protein localization, while Western blotting is used to measure SMN content in vitro. Quantitative real-time PCR is also carried out to quantify SMNl copy number,

[00103] Primary fibroblasts are expected to show partial to complete rescue of SMN protein and mRNA content when treated with exosomal SMNl mRNA.

Example 4 - Treatment of SMN deficiency in vivo with mRNA-loaded Exosomes

[00104] Four SMA breeder mice (SMN2+/+; SmnA7+/+ Smn+/-; possessing no overt phenotype and are hence used as breeders), were obtained from Jackson Laboratories (Maine, USA) to generate SMNA7 SMA (SMN2+/+; SmnA7+/+; Smn-/-; expressing the SMNA7 transcript and possess a phenotype similar to that of the human SMA disease) and non-SMA mice (SMN2+/+; SmnA7+/+; Smn+/+; possessing a wildtype SMN protein and hence used as experimental control). 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. [00105] A 7- week intravenous treatment of SMNA7 SMA mice with SMN 1 mRNA-loaded unmodified exosomes is conducted. Exosomes are loaded with an mRNA dose equivalent to delivery of -100-150 ng mRNA, in 10 ug of total exosomal proteins, isolated and prepared as described in Example 1.

[00106] Treatment of SMNA7 SMA mice with SMN1 mRNA-loaded exosomes is expected to restore SMN content in sciatic nerve, brain, spinal cord and skeletal muscle, to the levels seen in non-SMNl mice. Empty exosomes, naked SMN J mRNA and SMN1 mRNA-loaded exosomes are given to non-SMNl mice as controls.

Example 5 - Exosome Isolation using PEG-based method

[00107] Exosomes were isolated from various human and other mammalian biological samples as follows.

[00108] Blood samples were collected from healthy human subjects using red top serum collection tubes (e.g. BD, Ref #367812) and blue top plasma collection tubes containing sodium citrate (e.g. BD, Ref #369714) for serum and plasma isolations, respectively. For serum isolation, blood was allowed to clot for 1 hour at room temperature followed by centrifugation at 2,000x g for 15 min at 4°C. For plasma isolation, blood was spun down immediately after collection at 2,000x g for 15 min at 4°C. Plasma and serum was similarly collected from C57B1/6J mice and Sprague Dawley rats. Exosomes were then isolated from these samples, as well as from bovine whole milk (Natrel fine-filtered 3.25% milk) and cells in culture (e.g. CHO cells). From this point onwards, all exosome sources were treated the same.

[00109] Serum, plasma and milk were spun at 2000x g for 15 min at 4°C. The supernatant from the first centrifugation was spun at 2000x g for 60 min at 4°C to pellet debris. The supernatant was then spun at 15,000x g for 60 min at 4°C. The resulting supernatant was then filtered through a 45 μηι filter (Millipore, cat. # SLHV033RS), followed by filtration through a 0.22 μηι syringe filter (Millipore, cat. # SLGP0334B). The centrifugation and filtering steps have been determined to be optional steps. The filtered supernatant was then added to an equal volume of 16% PEG 6000 (Sigma, cat. # 81253) and 500mM NaCI in PBS (Bioshop, cat. # SOD002), mixed by inversion or gentle pipetting and incubated for 30 min at 4°C. The filtrate-PEG (8 %) solution was then spun at ΙΟ,ΟΟΟ^χ g for 0 min at 4°C to pellet the exosomes. The supernatant was discarded and the pellet was solubilized in 600uL of 0.5M trehalose (Sigma, cat. # TO 167) in PBS by gentle pipetting or on a mechanical plate rocker for 30 min at 4°C. Exosomes were further purified by applying the exosome-containing solution to centrifugation between 15,000x g - 150,000x g for 1 hr at 4°C. The resulting supernatant containing purified exosomes was then collected. A BCA assay (Pierce™) was used to determine exosome yield of between 5-1 Omg of exosomal protein per lmL of serum used. Transmission electron microscopy was performed on exosome solutions confirming the isolation of exosomes in the size range of 20-140 nm in diameter. The size distribution profile of exosomes isolated using the present PEG- based method was then measured using a Beckman DelsaMax dynamic light scattering analyzer, showing that the majority of particles in these solutions were within the 20-140 nm size range with minimal contamination outside of this exosome size range. Exosomal purity was further exemplified by performing Western blots with the canonical exosome markers CD9, CD63, CD81 and TSG101. Both the supernatant and pellet fractions of exosome solutions isolated from mouse serum and plasma samples using the PEG- based isolation method (and a final ultracentrifugation step) demonstrated robust expression of these markers confirming the presence of exosomes. The purity of exosomes was also determined by performing a Ponceau S stain, a widely used indicator for the presence of protein bands during Western blotting.

[00110] Relevant portions of references referred to herein are incorporated by reference.

[001 1 1] While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

1. Exosomes which are genetically modified to incorporate a functional neuropeptide or nucleic acid encoding a functional neuropeptide or precursor thereof

2. The exosomes of claim 1 , essentially free from particles having a diameter less than 20 nm or greater than 140 nm.

3. The exosomes of claim 1, wherein the neuropeptide or nucleic acid encoding the neuropeptide is exogenous.

4. The exosomes of claim 1, which are mammalian exosomes.

5. The exosomes of claim 1, wherein the nucleic acid is a gene selected from the group consisting of survival of motor neuron 1, telomeric (SMNl), vaccinia related kinase 1 (VRK1), exosome component 3 (EXOSC3), exosome component 8 (EXOSC8), immunoglobulin mu binding protein 2 (IGHMBP2), DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2), pleckstrin homology and RhoGEF domain containing G5 (PLE HG5), ubiquitin like modifier activating enzyme 1 (UBA1), ATPase copper transporting alpha (ATP7A), LAS 1 -like, ribosome biogenesis factor (LASIL), heat shock protein family B (small) member 1 (HSPBl), histidine triad nucleotide binding protein 1 (HI T1), ALS2, alsin Rho guanine nucleotide exchange factor (ALS2) , spastic paraplegia 11 (autosomal recessive) (SPG11), optineurin (OPTN), sigma non- opioid intracellular receptor 1 (SIGMAR1), solute carrier family 52 (riboflavin transporter), member 3 (SLC52A3), chromosome 12 open reading frame 65 (cl2orf65), spastic paraplegia 7 (pure and complicated autosomal recessive) (SPG7) , ER lipid raft associated 2 (ERLTN2), proteolipid protein 1 (PLP1), LI cell adhesion molecule (L1CAM), solute carrier family 16, member 2 (thyroid hormone transporter) (SLC16A2), adenosine monophosphate deaminase 2 (AMPD2), adaptor related protein complex 5 zeta 1 subunit (AP5Z1), ADP ribosylation factor like GTPase 6 interacting protein 1 (ARL6IP1), arylsulfatase family member I (ARSI), chromosome 10 open reading frame 2 (C10orf2), chaperonin containing TCP1 subunit 5 (CCT5), cytochrome P450 family 2 subfamily U member 1 (CYP2U1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), DDHD domain containing 1 (DDHD1), DDHD domain containing 2 (DDHD2), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), ER lipid raft associated 1 (ERLI l)j fatty acid 2-hydroxylase (FA2H), fibronectin leucine rich transmembrane protein 1 (FLRT1), gap junction protein gamma 2 (GJC2), kinesin family member 1A (KIF1A), kinesin family member 1C (KIF1C), methionyl-tRNA synthetase (MARS), 5'-nucIeotidase, cytosolic II (NT5C2), post-GPI attachment to proteins 1 (PGAP1), phospholipase A2 group VI (PLA2G6), patatin like phospholipase domain containing 6 (PNPLA6), polymerase (DNA directed), gamma (POLG), RAB3 GTPase activating non-catalytic protein subunit 2 (RAB3GAP2), sacsin molecular chaperone (SACS), ganglioside induced differentiation associated protein 1 (GDAP1), myotubularin related protein 2 (MTMR2), SET binding factor 1 (SBF1), SET binding factor 2 (SBF2), SH3 domain and tetratricopeptide repeats 2 (SH3TC2), N-myc downstream regulated 1 (NDRG1), early growth response 2 (EGR2), periaxin (PRX), hexokinase 1 (HK1), FYVE, RhoGEF and PH domain containing 4 (FGD4), FIG4 phosphoinositide 5-phosphatase (FIG4), surfeit 1 (SURFl), peripheral myelin protein 22 (PMP22), N-acylsphingosine amidohydrolase (acid ceramidase) 1 (ASAH1), phosphomannomutase 2 (PMM2), peroxisomal biogenesis factor 1 (PEX1), peroxisomal biogenesis factor 7 (PEX7), abhydrolase domain containing 12 (ABHD12), DnaJ heat shock protein family (Hsp40) member C3 (DNAJC3), phytanoyl-CoA 2-hydroxylase (PHYH), amin A/C (LMNA), mediator complex subunit 25 (MED25), leucine rich repeat and sterile alpha motif containing 1 (LRSAM1), tripartite motif containing 2 (TRIM2), polynucleotide kinase 3 '-phosphatase (PNKP), KIF1 A, solute carrier family 12 (potassium/chloride transporter), member 6 (SLC12A6), SCYl-like, kinase-like 1 (SCYL1), tyrosyl-DNA phosphodiesterase 1 (TDP1), PLA2G6, mitofusin 2 (MFN2), receptor accessory protein 1 (REEPl), neurofilament, light polypeptide (NEFL), gigaxonin (GAN), solute carrier family 25 member 46 (SLC25A46), gap junction protein beta 1 (GJB1), pyruvate dehydrogenase kinase 3 (PD 3), apoptosis inducing factor, mitochondria associated 1 (AIFM1), phospho ibosyl pyrophosphate synthetase 1 (PRPS1), WNK lysine deficient protein kinase 1 (WINK1), family with sequence similarity 134 member B (FAM134B), sodium voltage-gated channel alpha subunit 9 (SCN9A), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein (IKBKAP), neurotrophic tyrosine kinase, receptor, type 1 (NTRKl ), nerve growth factor (beta polypeptide) (NGF), dystonin (DST), PR domain 12 (PRDM12), clathrin heavy chain like 1 (CLTCL1), feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), tectonin beta-propeller repeat containing 2 (TECPR2) and combinations thereof.

6. The exosomes of claim 1 , further modified to incorporate or express a target-specific fusion product comprising a neuronal targeting sequence linked to an exosomal membrane marker.

7. The exosomes of claim 6, 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.

8. The exosomes of claim 6, wherein the neuronal targeting sequence is selected from the group consisting of myelin-associated glycoprotein, kinesin-like protein 1A, synthaxinl, synaptosomal-associated protein 25kDa, synaptobrevin and fragments thereof.

9. A composition comprising genetically modified exosomes as defined in claim 1 combined with a pharmaceutically acceptable carrier.

10. The composition of claim 9, comprising exosomal protein in an amount of about 100-2000 &

11. A method of treating a neuropathy in a mammal comprising administering to the mammal a composition comprising exosomes which are genetically modified to incorporate a functional neuropeptide or nucleic acid encoding the neuropeptide.

12. The method of claim 11 , wherein the neuropathy is a recessive neuronopathy or peripheral neuropathy selected from the group consisting of spinal muscular atrophy (SMA) type 0, SMA type 1, SMA type 2, SMA type 3, SMA type 4, SMA with pontocerebellar hypoplasia (PCH), distal SMA, Distal Hereditary Neuropathy (HMN), amyotrophic lateral sclerosis (ALS), hereditary spastic paraparesis (HSP), hereditary motor and sensory neuropathies (HMSN) and hereditary sensory and autonomic neuropathy (HSAN).

13. The method of claim 1 1, wherein the nucleic acid encoding the neuropeptide is selected from the group consisting of survival of motor neuron 1 , telomeric (SMN1), vaccinia related kinase 1 (VRK1), exosome component 3 (EXOSC3), exosome component 8 (EXOSC8), immunoglobulin mu binding protein 2 (IGHMBP2), DnaJ heat shock protein family (Hsp40) member B2 (DNAJB2), pleckstrin homology and RhoGEF domain containing G5 (PLEKHG5), ubiquitm like modifier activating enzyme 1 (UBA1), ATPase copper transporting alpha (ATP7A), LAS 1 -like, ribosome biogenesis factor (LAS1L), heat shock protein family B (small) member 1 (HSPB1), histidine triad nucleotide binding protein 1 (HI T1), ALS2, alsin Rho guanine nucleotide exchange factor (ALS2) , spastic paraplegia 11 (autosomal recessive) (SPG11), optineurin (OPTN), sigma non-opioid intracellular receptor 1 (SIGMAR1), solute carrier family 52 (riboflavin transporter), member 3 (SLC52A3) , chromosome 12 open reading frame 65 (cl2orf65), spastic paraplegia 7 (pure and complicated autosomal recessive) (SPG7) , ER lipid raft associated 2 (ERLIN2), proteolipid protein 1 (PLP1), LI cell adhesion molecule (L1CAM), solute carrier family 16, member 2 (thyroid hormone transporter) (SLC16A2), adenosine monophosphate deaminase 2 (AMPD2), adaptor related protein complex 5 zeta 1 subunit (AP5Z1), ADP ribosylation factor like GTPase 6 interacting protein 1 (ARL6IP1), arylsulfatase family member I (ARSI), chromosome 10 open reading frame 2 (C10orf2), chaperonin containing TCP1 subunit 5 (CCT5), cytochrome P450 family 2 subfamily U member 1 (CYP2U1), cytochiome P450 family 7 subfamily B member 1 (CYP7B1), DDHD domain containing 1 (DDHDl), DDHD domain containing 2 (DDHD2), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), ER lipid raft associated 1 (ERLIN1), fatty acid 2-hydroxylase (FA2H), fibronectin leucine rich transmembrane protein 1 (FLRT1), gap junction protein gamma 2 (GJC2), kinesin family member 1A (KIF1A), kinesin family member 1C (KIF1C), methionyl-tRNA synthetase (MARS), 5'- nucleotidase, cytosolic II (NT5C2), post-GPI attachment to proteins 1 (PGAP1), phospholipase A2 group VI (PLA2G6), patatin like phospholipase domain containing 6 (PNPLA6), polymerase (DNA directed), gamma (POLG), RAB3 GTPase activating non-catalytic protein subunit 2 (RAB3GAP2), sacsin molecular chaperone (SACS), ganglioside induced differentiation associated protein 1 (GDAP1), myotubularin related protein 2 (MTMR2), SET binding factor 1 (SBF1), SET binding factor 2 (SBF2), SH3 domain and tetrat icopeptide repeats 2 (SH3TC2), N- myc downstream regulated 1 ( DRG1), early growth response 2 (EGR2), periaxin (PRX), hexokinase 1 (H 1), FYVE, RhoGEF and PH domain containing 4 (FGD4), FIG4 phosphoinositide 5-phosphatase (FIG4), surfeit ί (SURF1), peripheral myelin protein 22 (PMP22), N-acylsphingosine amidohydrolase (acid ceramidase) 1 (ASAH1), phosphomannomutase 2 (PMM2), peroxisomal biogenesis factor 1 (PEX1), peroxisomal biogenesis factor 7 (PEX7), abhydrolase domain containing 12 (ABHD12), DnaJ heat shock protein family (Hsp40) member C3 (DNAJC3), phytanoyl-CoA 2-hydroxylase (PHYH), amin A/C (LMNA), mediator complex subunit 25 (MED25), leucine rich repeat and sterile alpha motif containing 1 (L SAM1), tripartite motif containing 2 (TRIM2), polynucleotide kinase 3'- phosphatase (PNKP), K1F1 A, solute carrier family 12 (potassium/chloride transporter), member 6 (SLC12A6), SCYMike, kinase-like 1 (SCYL1), tyrosyl-DNA phosphodiesterase 1 (TDP1), PLA2G6, mitofusin 2 (MFN2), receptor accessory protein 1 (REEP1), neurofilament, light polypeptide (NEFL), gigaxonin (GAN), solute carrier family 25 member 46 (SLC25A46), gap junction protein beta 1 (GJB1), pyruvate dehydrogenase kinase 3 (PDK3), apoptosis inducing factor, mitochondria associated 1 (AIFM1), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), WN lysine deficient protein kinase 1 (WINK1), family with sequence similarity 134 member B (FAM134B), sodium voltage-gated channel alpha subunit 9 (SCN9A), inhibitor of kappa light polypeptide gene enhancer in B-cells₅ kinase complex-associated protein (IKBKAP), neurotrophic tyrosine kinase, receptor, type 1 (NTRK1), nerve growth factor (beta polypeptide) (NGF), dystonin (DST), PR domain 12 (PRDM12), clathrin heavy chain like 1 (CLTCL1), feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), tectonin beta-propeller repeat containing 2 (TECPR2) and combinations thereof.

14. The method of claim 12, wherein the recessive neuronopathy is spinal muscular atrophy and the protein is survival of motor neuron.

15. The method of claim 1 1, wherein a dosage of exosomes sufficient to deliver an amount of nucleic acid to yield about 0.1 ng/kg to about 100 ug kg of the neuropeptide is administered to the mammal.

16. A method of increasing the amount or activity of a neuropeptide in a mammal, comprising administering to the mammal a composition comprising exosomes that are genetically modified to incorporate a functional neuropeptide or nucleic acid encoding the neuropeptide.

17. The method of claim 16, wherein the exosomes are essentially free from particles having a diameter less than 20 nm or greater than 140 nm.

18. The method of claim 16, wherein the neuropeptide or nucleic acid is exogenous.

19. The method of claim 16, wherein the exosomes are isolated from a biological sample using a method comprising the following steps: i) optionally 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) optionally subjecting the supernatant from step i) to centrifugation to remove microvesicles and apoptotic bodies therefrom; iii) optionally microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) combining the microfiltered supernatant from step iii) with a polyethylene glycol (PEG) solution to precipitate the exosomes and subjecting the solution to at least one round of centrifugation to obtain an exosome pellet; and v) re-suspending the exosome pellet from step iv) in a trehalose solution and conducting an optional centrifugation step to remove vesicles having a diameter of greater than 140 nm from the solution.