Scalable Production of Standardized Extracellular Vesicles, Extracellular Vesicle Preparations and Uses Thereof

Abstract

Preparations comprising an enriched population of extracellular vesicles (nEVs) having a negatively charged surface, and that are CD81+ and CD9−, are provided. Improved processes and methods for producing an enriched population of nEVs from non-murine cells, especially human origin cells and/or tissues, are disclosed. Therapeutic methods for using the preparations, including for reducing brain inflammation and treatment of various pathologies associated with brain inflammation, such as by intravenous or intranasal administration, are also described. Methods and preparations for reducing brain inflammation associated with traumatic brain injury (TBI) are also disclosed. A method for treating a patient having suffered a mild traumatic injury (mTMI), or concussion, such as a sports-related head injury, is also disclosed. The nEVs are also demonstrated to reduce the expression level of IL-Iβ in brain tissue of an animal having had traumatic brain injury. Methods for improving cognitive function and performance in animals after a traumatic brain injury is also demonstrated using the preparations of nEVs disclosed herein.

What is claimed is: 
     
         1 . A pharmaceutical preparation comprising an enriched population of selected extracellular vesicles having a negatively charged surface, said selected negative extracellular vesicles (n-EVs) lacking a CD9 surface epitope (CD9 − ). 
     
     
         2 . The pharmaceutical preparation of  claim 1  wherein the selected extracellular vesicles having a mean size of about 80 nm to about 250 nm. 
     
     
         3 . The pharmaceutical preparation of  claim 1  suitable for intravenous administration or intranasal administration. 
     
     
         4 . The pharmaceutical preparation of  claim 1  wherein the selected extracellular vesicles comprise at least about 50% by volume of the pharmaceutical preparation. 
     
     
         5 . The pharmaceutical preparation of  claim 1  wherein the selected extracellular vesicles lack any combination of two or more of the surface epitopes selected from the group consisting essentially of: CD29, CD44, CD49c, CD49f, CD59, CD73, CD90, CD105, and CD166. 
     
     
         6 . The pharmaceutical preparation of  claim 1  wherein the preparation comprises mature TSG-G mRNA, cytokines, chemokines micro-RNA's and mitochondria. 
     
     
         7 . A pharmaceutically acceptable intravenous preparation comprising the pharmaceutical preparation of  claim 1  and a pharmaceutically acceptable carrier solution. 
     
     
         8 . A scalable method for producing an enriched population of negatively charged extracellular vesicles, said method comprising:
 (a) culturing a population of undifferentiated human cells under appropriate conditions suitable for inducing differentiation of said cells to mesenchymal stem cells;   (b) culturing said induced population of human cells for between about 6 hours and about 48 hours in a supplemented, protein-free culture medium that prevents cell aggregation of cells synthesizing TSG-6, to produce a conditioned culture medium;   (c) collecting the conditioned media and removing cellular debris from said conditioned culture medium;   (d) passing the conditioned culture medium over an anion exchange column to provide a population of extracellular vesicles having a negative surface charge (n-EVs) attached to said column;   (e) removing the population of n-EVs from the column; and   (f) selecting from said population of n-EVs a subset of n-EVs having activity for suppressing neuroinflammation and having a surface epitope that is absent CD9 (CD9).   
     
     
         9 . The method of  claim 8  wherein the anion exchange column is a resin anion exchange column. 
     
     
         10 . The method of  claim 8  wherein the culture medium comprises a protein-free, CD-CHO culture medium supplemented with hypoxanthine, thymidine, glutamine, glucose, essential amino acids and vitamins. 
     
     
         11 . The method of  claim 8  wherein the human mesenchymal stem cells are derived from human bone marrow. 
     
     
         12 . A method for reducing brain inflammation comprising:
 (a) administering a pharmaceutically acceptable preparation comprising an enriched preparation of negatively charged n-EVs and absent a CD9 epitope (CD9 − ) to an animal at risk of having or having inflammation attendant traumatic brain injury; and   (b) observing a reduction in brain inflammation in the animal,   
       wherein a reduction in brain inflammation is correlated with a return to consciousness, improved mobility, or other physically manifested improvement. 
     
     
         13 . The method of  claim 12  wherein the brain inflammation is attendant traumatic brain injury. 
     
     
         14 . A method of improving cognitive function in an animal having traumatic brain injury comprising administering to the animal a therapeutically effective amount of the pharmaceutical preparation of  claim 7 . 
     
     
         15 . The method of  claim 14  wherein the pharmaceutical preparation is administered intravenously (i.v.). 
     
     
         16 . A method for improving pattern separation function and spatial learning ability in an animal subsequent a traumatic brain injury (TBI) comprising:
 (a) providing a base-line separation function and spatial learning ability of an animal having had a traumatic brain injury (TBI) to provide a base-line assessment measurement;   (b) administering a pharmaceutically acceptable preparation comprising an enriched preparation of negatively charged n-EVs that are absent a CD9 epitope (CD9 − ) to an animal at risk of having or having inflammation attendant traumatic brain injury to provide a treated animal;   (c) assessing the treated animal for separation function and spatial learning ability to provide post-treatment assessment measurements, and comparing the post-treatment assessment measurements to the base-line separation function and spatial learning assessment measurements; and   (d) providing an animal having improved pattern separation function and spatial learning ability where the post-treatment assessment measurements are improved compared to the base-line assessment measurements.   
     
     
         17 . The method of  claim 16  wherein the preparation of n-EVs administered comprises about 30 μg of protein and 15×10 9  EVs by weight of the preparation 
     
     
         18 . An enriched population of pharmacologically active n-EVs that have a CD9 −  surface epitope, prepared by a method comprising the steps of:
 (a) culturing a population of undifferentiated human cells under appropriate conditions suitable for inducing differentiation of said cells to mesenchymal stem cells; 
 (b) culturing said induced population of human cells for between about 6 hours and about 48 hours in a supplemented, protein-free culture medium that prevents cell aggregation of cells synthesizing TSG-6, to produce a conditioned culture medium; 
 (c) collecting the conditioned media and removing cellular debris from said conditioned culture medium; 
 (d) passing the conditioned culture medium over an anion exchange column to provide a population of extracellular vesicles having a negative surface charge (n-EVs) attached to said column; 
 (e) removing the population of n-EVs from the column; and 
 (f) selecting from said population of n-EVs a subset of n-EVs having activity for suppressing neuroinflammation and having a surface epitope that is absent CD9 (CD9 − ) to provide an enriched population of pharmacologically active n-EVs having a CD9 −  surface epitope. 
 
     
     
         19 . A method for producing an enriched preparation of negatively charged extracellular vesicles (n-EVs) having brain anti-inflammatory pharmacological activity in vivo, comprising:
 (a) culturing a population of human mesenchymal stem cells for between about 6 hours and about 48 hours in a supplemented, protein-free culture medium that prevents cell aggregation of cells synthesizing TSG-6, to produce a conditioned culture medium;   (b) collecting the conditioned media and removing cellular debris from said conditioned culture medium;   (c) passing the conditioned culture medium over an anion exchange column to provide a population of extracellular vesicles having a negative surface charge (n-EVs) attached to said column;   (d) removing the population of n-EVs from the column in an eluate from the column; and   (e) selecting from said population of n-EVs a subset of n-EVs having activity for suppressing brain tissue neuroinflammation and having a surface epitope that is absent CD9 (CD9 − ) to provide an enriched population of pharmacologically active n-EVs having a CD9 −  surface epitope.   
     
     
         20 . The method of  claim 19  wherein the method comprises an additional step of passing the eluate of step (c) over a second anion exchange column. 
     
     
         21 . A method for treating concussion in a human subject comprising:
 (a) identifying a person who has experienced a concussion;   (b) administering a pharmaceutically acceptable preparation comprising an enriched preparation of negatively charged, n-EVs that are absent a CD9 epitope (CD9 − ) to the person identified to have had a concussion; and   (c) assessing the response of the person to the administered preparation,   
       wherein a positive response to the administered preparation is identified in the treated person that evidences a state of regained consciousness. 
     
     
         22 . The method of  claim 21  wherein the concussion is incident a sports-related head injury.