Therapeutic delivery vesicles

Abstract

The present invention pertains to inter alia methods for purifying extracellular vesicles including exposing a sample comprising at least one extracellular vesicle to ultrafiltration; and exposing the sample following the ultrafiltration in step (i) to size exclusion liquid chromatography.

What is claimed is: 
     
         1 . A method for purifying extracellular vesicles comprising:
 (i) exposing a sample comprising at least one extracellular vesicle to ultrafiltration; and   (ii) exposing the sample following the ultrafiltration in step (i) to size exclusion liquid chromatography.   
     
     
         2 . A method of  claim 1 , wherein at least one filter is used during ultrafiltration. 
     
     
         3 . A method of  claim 2 , wherein the at least one filter comprises a molecular weight cut-off of 100 kDa. 
     
     
         4 . A method of  claim 2 , wherein the at least one filter comprises a molecular weight cut-off of 200 kDa. 
     
     
         5 . A method of  claim 2 , wherein the at least one filter comprises a molecular weight cut-off of 500 kDa. 
     
     
         6 . A method of  claim 1 , wherein at least two different filters are used during ultrafiltration and wherein the at least two different filters have the same molecular weight cut-off. 
     
     
         7 . A method of  claim 1 , wherein at least two different filters are used during ultrafiltration and wherein the at least two different filters have a different molecular weight cut-off. 
     
     
         8 . A method of  claim 1 , further comprising prior to step (i) exposing the sample to stress-inducing conditions. 
     
     
         9 . A method of  claim 8 , wherein the stress-inducing conditions are oxygen deprivation, serum starvation, or a combination thereof. 
     
     
         10 . A method of  claim 1 , further comprising prior to step (i) exposing the sample to autophagy-inhibiting substances. 
     
     
         11 . A method of  claim 1 , wherein the extracellular vesicles purified using ultrafiltration followed by size exclusion liquid chromatography comprise less protein contamination than extracellular vesicles purified using ultrafiltration alone. 
     
     
         12 . A method of  claim 1 , wherein the extracellular vesicles purified using ultrafiltration followed by size exclusion liquid chromatography comprise less vesicle distortion and improved vesicle integrity than extracellular vesicles purified using ultrafiltration alone. 
     
     
         13 . A method for purifying extracellular vesicles, comprising:
 (i) introducing at least one polynucleotide construct encoding at least one polypeptide into a cell capable of producing extracellular vesicles;   (ii) collecting the extracellular vesicles produced by the cell of step (i); and   (iii) purifying the extracellular vesicles collected in step (ii) using ultrafiltration followed by size exclusion liquid chromatography.   
     
     
         14 . A method of  claim 13 , wherein at least one filter is used during ultrafiltration. 
     
     
         15 . A method of  claim 14 , wherein the at least one filter comprises a molecular weight cut-off of 100 kDa. 
     
     
         16 . A method of  claim 14 , wherein the at least one filter comprises a molecular weight cut-off of 200 kDa. 
     
     
         17 . A method of  claim 14 , wherein the at least one filter comprises a molecular weight cut-off of 500 kDa. 
     
     
         18 . A method of  claim 13 , wherein at least two different filters are used during ultrafiltration and wherein the at least two different filters have the same molecular weight cut-off. 
     
     
         19 . A method of  claim 13 , wherein at least two different filters are used during ultrafiltration and wherein the at least two different filters have a different molecular weight cut-off. 
     
     
         20 . A method of  claim 13 , further comprising prior to step (ii) exposing the cell to stress-inducing conditions. 
     
     
         21 . A method of  claim 20 , wherein the stress-inducing conditions are oxygen deprivation, serum starvation, or a combination thereof. 
     
     
         22 . A method of  claim 13 , further comprising prior to step (ii) exposing the cell to autophagy-inhibiting substances. 
     
     
         23 . A method of  claim 13 , wherein the extracellular vesicles purified using ultrafiltration followed by size exclusion liquid chromatography comprise less protein contamination than extracellular vesicles purified using ultrafiltration alone. 
     
     
         24 . A method of  claim 13 , wherein the extracellular vesicles purified using ultrafiltration followed by size exclusion liquid chromatography comprise less vesicle distortion and improved vesicle integrity than extracellular vesicles purified using ultrafiltration alone.