Delivery of therapeutic agent

Abstract

A method of producing nanovesicles comprising an oligonucleotide inhibitor to an oncogene or a proto-oncogene or the gene product thereof, said method comprises a) introducing a DNA sequence encoding an oligonucleotide capable of inhibiting a human oncogenic or proto-oncogenic transcription factor, into a mammalian cell; b) allowing the cell to express said inhibitor oligonucleotide; and c) obtaining nanovesicles containing said inhibitor oligonucleotide from said cell. Nanovesicles produced by the claimed method can be effectively and specifically targeted to e.g. cancer cells to deliver the inhibitor oligonucleotide.

1 . A method of producing nanovesicles comprising an inhibitor oligonucleotide to an oncogene or a proto-oncogene or the gene product thereof, the method comprising a) introducing a DNA sequence encoding an oligonucleotide capable of inhibiting a human oncogenic or proto-oncogenic transcription factor into a mammalian cell;
 b) maintaining the cell under conditions allowing the cell to express the inhibitor oligonucleotide; and   c) obtaining nanovesicles containing the inhibitor oligonucleotide from the cell.   
     
     
         2 . The method according to  claim 1 , wherein the inhibitor oligonucleotide is an inhibitor to a human MYC gene or gene product thereof. 
     
     
         3 . The method according to  claim 1 , wherein the inhibitor oligonucleotide is an RNAi molecule. 
     
     
         4 . (canceled) 
     
     
         5 . The method according to  claim 1 , wherein the cell expresses a targeting molecule on the cell surface. 
     
     
         6 . The method according to  claim 1 , wherein the method comprises, prior to step a) or simultaneously with step a), introducing into the cell a DNA sequence coding for a targeting molecule to be expressed on the cell surface. 
     
     
         7 . The method according to  claim 5 , wherein the targeting molecule is a ligand specifically binding to a surface protein that is overexpressed on cancer cells. 
     
     
         8 . The method according to  claim 7 , wherein the targeting molecule is epidermal growth factor (EGF), melanocyte-stimulating hormone (MSH) or a variable region of an antibody directed against a surface protein that is overexpressed on cancer cells, or a fab-fragment of the antibody. 
     
     
         9 . The method according to  claim 1 , wherein the cell is a human cell. 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The method according to  claim 1 , performed in vitro or ex vivo. 
     
     
         13 . An isolated nanovesicle produced by the method according to  claim 1 . 
     
     
         14 . An isolated nanovesicle comprising an inhibitor oligonucleotide to a human oncogenic or proto-oncogenic transcription factor. 
     
     
         15 . The isolated nanovesicle according to  claim 14 , wherein the oncogenic or proto-oncogenic transcription factor is a member of a MYC gene or gene products family. 
     
     
         16 . The isolated nanovesicle according to  claim 14 , wherein the oncogene or proto-oncogene is a member of an E2F gene or gene products family. 
     
     
         17 . The isolated nanovesicle according to  claim 14 , wherein the oligonucleotide is an RNAi molecule. 
     
     
         18 . The isolated nanovesicle according to claim_17, wherein the RNAi molecule is shRNA or siRNA. 
     
     
         19 . The isolated nanovesicle according to  claim 14 , which is an artificial nanovesicle produced by serial extrusions of cells through microfilters and/or nanofilters. 
     
     
         20 . A human cell transfected or transduced with a genetic construct comprising a gene whose gene product functionally replaces a human oncogenic or proto-oncogenic transcription factor naturally expressed by human cell, wherein the human cell further contains a DNA sequence encoding an oligonucleotide capable of inhibiting the human oncogenic or proto-oncogenic transcription factor. 
     
     
         21 . The human cell according to  claim 20 , wherein the human oncogenic or proto-oncogenic transcription factor is encoded by one member of a MYC gene family, and the genetic construct comprises another member of a MYC gene family. 
     
     
         24 . The human cell according to  claim 20 , which is a stem cell. 
     
     
         25 . A composition comprising a nanovesicle according to  claim 14  and a pharmaceutically acceptable carrier.