Delivery of therapeutic agent
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, said method comprising
(a) transfecting or transducing a mammalian cell with a construct comprising L-Myc genes;
(b) maintaining the cell under conditions allowing the cell to overexpress L-Myc;
c) introducing into the cell a DNA sequence encoding an oligonucleotide capable of inhibiting a human oncogenic or proto-oncogeneic transcription factor wherein said inhibitor does not inhibit L-Myc; and
(d) obtaining nanovesicles containing said inhibitor oligonucleotide from said cell.
2. A method according to claim 1 , wherein said inhibitor oligonucleotide is an inhibitor to a human MYC gene or gene product, preferably the c-Myc gene or gene product.
3. A method according to claim 1 , wherein said oligonucleotide is RNA, preferably an RNAi molecule.
4. A method according to claim 3 , wherein said DNA sequence encoding an oligonucleotide capable of inhibiting a human oncogenic or proto-oncogenic transcription factor is a DNA sequence selected from the group consisting of SEQ ID NO: 1-9.
5. A method according to claim 1 , wherein said cell expresses a targeting molecule on the cell surface.
6. A method according to claim 1 , wherein the method comprises, prior to step (c) or simultaneously with step (c), introducing a DNA sequence encoding a targeting molecule to be expressed on the cell surface.
7. A method according to claim 5 , wherein said targeting molecule is a ligand specifically binding to a surface protein that is overexpressed on cancer cells.
8. A method according to claim 7 , wherein said 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 said antibody.
9. A method according to claim 1 , wherein the cell is a human cell.
10. A method according to claim 1 , wherein step (c) comprises introducing a genetic construct comprising said DNA sequence coding for an oligonucleotide capable of inhibiting human c-Myc, operatively linked to an inducible promoter, and inducing expression by activation of said inducible promoter.
11. A method according to claim 1 , performed in vitro or ex vivo.
12. A method according to claim 3 , wherein said DNA sequence encoding an oligonucleotide capable of inhibiting a human oncogenic or proto-oncogenic transcription factor is a DNA sequence selected from the group consisting of SEQ ID NO: 185-196.