Extracellular vesicles derived from osteoblast lineage cells for therapeutic and diagnostic use

Abstract

The present invention relates to RANKL+ cellular vesicles isolated from osteoblastic lineage cells, optionally immortalised, their use for the therapeutic treatment of bone pathologies and for diagnostic purposes, and processes for the production of said vesicles.

1 - 21 . (canceled) 
     
     
         22 . A method of preparation of extracellular vesicles isolated from osteoblastic lineage cells, optionally wherein the cells are immortalised, wherein the method comprises the steps of:
 (a) culturing the cells; and   (b) collecting, by ultrafiltration and/or ultracentrifugation, the extracellular vesicles produced by the cultured cells.   
     
     
         23 . The method of  claim 22 , wherein step (a) is carried out in the presence of parathyroid hormone; and optionally wherein the cells are genetically manipulated to overexpress RANKL. 
     
     
         24 . The method according to  claim 22 , further comprising step (c), ultrafiltration with 0.22 μm filters. 
     
     
         25 . (canceled) 
     
     
         26 . A method for loading extracellular vesicles isolated from osteoblastic lineage cells, optionally immortalised, with one or more therapeutic agents or with one or more contrast agents, wherein the method comprises the steps of:
 (a) incubating the vesicles, and/or osteoblastic lineage cells, optionally immortalised in a culture media containing one or more therapeutic agents or one or more contrast agents; and   (b) collecting the loaded vesicles by ultracentrifugation, by filtration, by ultrafiltration, by column filtration, by ultracentrifugation on sucrose gradient, or by separation with immunomagnetic methods.   
     
     
         27 . The method according to  claim 26 , wherein step (a) is preceded by step (y), electroporation. 
     
     
         28 . The method according to  claim 26 , wherein the one or more therapeutic agents are selected from the group consisting of chemotherapeutic agents, siRNAs, miRNAs, anti-miRNAs, constructs for transient transformation, antibiotics, pharmacological molecules, and antibiotics or mixtures thereof. 
     
     
         29 . The method according to  claim 26 , wherein the one or more contrast agents is an agent used in an imaging diagnostic technique. 
     
     
         30 . The method according to  claim 29 , wherein the imaging diagnostic technique is a radiologic technique with X rays, scintigraphy, or nuclear magnetic resonance. 
     
     
         31 . The method according to  claim 30 , wherein the contrast agent used in the imaging diagnostic technique is selected from the group consisting of an X-ray opaque compound, a radioactive isotope, and a contrast agent for nuclear magnetic resonance. 
     
     
         32 . The method according to  claim 31 , wherein the X-ray opaque compound is an iodate compound. 
     
     
         33 . The method according to  claim 31 , wherein the radioactive isotope is selected from the group consisting of 99m technetium, iodine 131, thallium 201, and iodine 125. 
     
     
         34 . The method according to  claim 31 , wherein the contrast agent for nuclear magnetic resonance contains gadolinium chelated compounds. 
     
     
         35 . A method for loading extracellular vesicles isolated from osteoblastic lineage cells with one or more therapeutic agents, the method comprising the steps of:
 (a) transforming osteoblastic lineage cells, optionally wherein the cells are immortalised cells, to express a therapeutic agent of interest;   (b) culturing said osteoblastic lineage cells transformed in step (a); and   (c) collecting the loaded vesicles by ultracentrifugation, by filtration, by ultrafiltration, by column filtration, by ultracentrifugation on sucrose gradient, or by separation with immunomagnetic methods the vesicles thus loaded.   
     
     
         36 . The method according to  claim 26 , the method further comprising step (c), ultrafiltration with 0.22 μm filters. 
     
     
         37 - 39 . (canceled) 
     
     
         40 . The method of  claim 22 , further comprising loading the extracellular vesicles collected in step (b) with one or more therapeutic agents or with one or more contrast agents. 
     
     
         41 . A method of treatment for a bone pathology comprising administration to a subject in need thereof one or more therapeutically effective doses of extracellular vesicles isolated from osteoblastic lineage cells, optionally wherein the cells are immortalised cells, wherein the vesicles comprise RANKL (Receptor Activator of NF-KB Ligand) on one or more membranes of the vesicles, wherein the vesicles further comprise suitable therapeutic agents. 
     
     
         42 . The method of  claim 41 , wherein the bone pathology is selected from primitive and secondary bone tumours. 
     
     
         43 . The method of  claim 41 , wherein the bone pathology is a bone metabolic alteration. 
     
     
         44 . The method of  claim 41 , further comprising a diagnostic step comprising imaging the bone pathology using the extracellular vesicles, wherein the vesicles comprise suitable contrast agents; and
 wherein the diagnostic step occurs before administration of the one or more therapeutically effective doses.