Bibliographic Details
| Title: |
SCLEROPROTEIN OF AN ADENO-ASSOCIATED VIRUS WITH MODIFIED CHROMATOGRAPHIC PROPERTIES, THE PRODUCTION THEREOF AND USE OF THE SAME |
| Document Number: |
20090246854 |
| Publication Date: |
October 1, 2009 |
| Appl. No: |
11/903662 |
| Application Filed: |
September 24, 2007 |
| Abstract: |
The invention relates to a scleroprotein of an adeno-associated virus which contains at least one mutation. Said mutation causes the chromatographic properties to be modified. The invention also relates to the production of said scleroprotein and the use thereof. |
| Inventors: |
Hallek, Michael (Koeln, DE); Girod, Anne (Jesenwang, DE); Ried, Martin (Starnberg, DE); Stolla, Christof (Basel, CH); Moebius, Ulrich (Gauting-Unterbrunn, DE) |
| Assignees: |
MEDIGENE AKTIENGESELLSCHAFT (Planegg Martinsried, DE) |
| Claim: |
1. A method for purifying adeno-associated virus (AAV) and/or AAV particles, said method comprising using an adeno-associated virus having a structural protein that comprises at least one mutation which brings about an alteration in the chromatographic properties of the virus. |
| Claim: |
2. The method as claimed in claim 1, characterized in that the alteration in the chromatographic properties makes an improvement in the purification possible, in particular a concentration of the virus, preferably of the virus particles, to higher titers, a purification to greater purity and/or a more efficient purification. |
| Claim: |
3. The method as claimed in claim 1, characterized in that the mutation brings about a negligible reduction in the infectivity of the virus. |
| Claim: |
4. The method as claimed in claim 1, characterized in that the mutated structural protein is capable of particle formation. |
| Claim: |
5. The method as claimed in claim 1, characterized in that the mutated structural protein is capable of particle formation. |
| Claim: |
6. The method as claimed in claim 1, characterized in that the structural protein is selected from mutated VP1, mutated VP2 and/or mutated VP3. |
| Claim: |
7. The method as claimed in claim 1, characterized in that the structural protein is derived from AAV1, AAV2, AAV3, AAV4, AAV5 and/or AAV6 and other AAV serotypes derived therefrom, in particular from AAV2. |
| Claim: |
8. The method as claimed in claim 1, characterized in that the mutation is a point mutation, a mutation of more than one amino acid, one or more deletion(s), in particular one or more insertion(s) or a combination of said modifications. |
| Claim: |
9. The method as claimed in claim 1, characterized in that amino acids of a functional sequence which are preferably suitable for affinity chromatography are inserted. |
| Claim: |
10. The method as claimed in claim 9, characterized in that the inserted amino acid sequence is selected from a ligand of a receptor or the receptor of a ligand, an antibody or part of an antibody, in particular an antibody epitope, an antigen or antigen epitope, a hormone, a hormone receptor, an enzyme, an enzyme substrate, a lectin, sugar-bearing amino acids, in particular from a histidine-rich peptide (His tag), a multiply charged peptide, glutathione S-transferase (GST tag), an Fc part of an antibody, an immunoglobulin-binding domain, for example protein A or protein G or a part thereof, a lecitin, a nucleic acid binding site, a heparin binding site, a specific ligand, a specific receptor, an integrin, a cytokine or a receptor binding domain of a cytokine, integrin or growth factor, single-chain antibodies which bind to a cell surface receptor, an antibody against cell surface structures, an epitope and/or an antibody-binding structure. |
| Claim: |
11. The method as claimed in claim 9, characterized in that a peptide which has the sequence QAGTFALRGDNPQG (SEQ ID NO: 1) is inserted into said structural protein. |
| Claim: |
12. The method as claimed in claim 1, characterized in that the structural protein comprises at least one other mutation. |
| Claim: |
13. The method in claimed in claim 12, characterized in that the other mutation(s) brings about an alteration in the infectivity of the virus. |
| Claim: |
14. The method as claimed in claim 12, characterized in that the other mutation(s) brings about a reduction in the antigenicity of the virus. |
| Claim: |
15. The method as claimed in claim 12, characterized in that the other mutation(s) is/are one or more deletions(s), one or more insertion (s) or a combination of said modifications. |
| Claim: |
16. The method as claimed in claim 15, characterized in that the insertion is a cell membrane receptor ligand, a Rep protein or peptide, an immunosuppressive protein or peptide and/or a protein or peptide with a signal for double strand synthesis of the foreign gene. |
| Claim: |
17. The method as claimed in claim 15, characterized in that the insertion is selected from an integrin, a cytokine or a receptor binding domain of a cytokine, integrin or growth factor, single-chain antibodies which bind to a cell surface receptor, an antibody against cell surface structures, an antibody-binding structure or an epitope. |
| Claim: |
18. The method as claimed in claim 1, characterized in that the mutation(s) is/are located on the virus surface. |
| Claim: |
19. The method as claimed in claim 1, characterized in that the mutation(s) is/are located at the N terminus of the structural protein. |
| Claim: |
20. The method as claimed in claim 1, characterized in that the mutation (s) is/are brought about by one or more insertions in the XhoI cleavage site of the VP1-encoding nucleic acid. |
| Claim: |
21. The method as claimed in claim 1, characterized in that the mutation(s) is/are brought about by one or more insertions in the BsrBI cleavage site of the VP1-encoding nucleic acid. |
| Claim: |
22. The method as claimed in claim 1, characterized in that the mutation (s) is/are brought about by one or more deletions between the BsrBI-HindIII cleavage sites of the VP1-encoding nucleic acid and one or more insertions. |
| Claim: |
23. The method as claimed in claim 1, characterized in that the mutation(s) is/are brought about by one or more deletions between the XhoI-XhoI cleavage sites of the VP1-encoding nucleic acid. |
| Claim: |
24. The method as claimed in claim 1, characterized in that the mutation(s) is/are brought about by one or more deletions between the BsrBI-HindIII cleavage sites of the VP1-encoding nucleic acid |
| Claim: |
25. The method as claimed in claim 1, characterized in that one or more insertions in VP3 is/are located before and/or after at least one amino acid in the sequence selected from YKQIS SQSGA (SEQ ID NO: 2), YLTLN NGSQA (SEQ ID NO: 3), YYLSR TNTPS (SEQ ID NO: 4), EEKFF PQSGV (SEQ ID NO: 5), NPVAT EQYGS (SEQ ID NO: 14), LQRGN RQAAT (SEQ ID NO: 8), NVDFT VDTNG (SEQ ID NO: 9). |
| Claim: |
26. The method as claimed in claim 1 characterized in that an AAV particle, in particular in the form of an AAV capsid, is purified. |
| Current U.S. Class: |
435236/000 |
| Current International Class: |
12; 12 |
| Accession Number: |
edspap.20090246854 |
| Database: |
USPTO Patent Applications |
