Inducible AAV Rep genes
The present invention relates to host cells comprising a nucleic acid encoding Adeno-associated virus (AAV) Rep proteins Rep78 and Rep68, wherein the internal AAV promoter p19 has been inactivated by one or more mutations that maintain the functionality of said Rep78 and Rep68 proteins. The present invention further relates to respective nucleic acids and vectors comprising the same, as well as respective methods for the production of AAV.
1. An isolated host cell comprising a nucleic acid encoding Adeno-associated virus (AAV) Rep proteins Rep78 and Rep68 operably linked to an inducible promoter, wherein the internal AAV promoter p19 comprises a substitution at position 818 from a guanine to an adenine wherein the position 818 is numbered according to a complete AAV2 genome of GenBank Accession number AF043303, whereby the internal AAV promoter p19 is inactivated and the functionality of said Rep78 and Rep68 proteins is maintained.
2. The isolated host cell according to claim 1 , wherein said nucleic acid comprises SEQ ID NO: 36.
3. The isolated host cell according to claim 1 , wherein said nucleic acid comprises SEQ ID NO: 45.
4. An isolated host cell, comprising a nucleic acid encoding Adeno-associated virus (AAV) Rep proteins Rep78 and Rep68 operably linked to an inducible promoter, wherein the internal AAV promoter p19 comprises a substitution at position 818 from a guanine to an adenine wherein the position 818 is numbered according to a complete AAV2 genome of GenBank Accession number AF043303, whereby the internal AAV promoter p19 is inactivated and the functionality of said Rep78 and said Rep68 proteins is maintained, wherein the nucleic acid also has at least 70% sequence identity to SEQ ID NO: 36.
5. The isolated host cell according to claim 1 , wherein said nucleic acid is stably integrated into the host cell genome or is comprised in a vector.
6. The isolated host cell according to claim 1 , further comprising a different nucleic acid encoding AAV Rep proteins Rep52 and Rep40 under the control of a heterologous, inducible promoter.
7. The isolated host cell according to claim 1 , selected from the group consisting of CAP cells, HEK293 cells, and Per.C6 cells.
8. A nucleic acid encoding Adeno-associated virus (AAV) Rep proteins Rep78 and Rep68 operably linked to an inducible promoter, wherein the internal AAV promoter p19 comprises a substitution at position 818 from a guanine to an adenine wherein the position 818 is numbered according to a complete AAV2 genome of GenBank Accession number AF043303, whereby the internal AAV promoter p19 is has been inactivated by one or more mutations that maintain and the functionality of said Rep78 and Rep68 proteins is maintained.
9. A method for the production of Adeno-associated virus (AAV), comprising the step of recombinantly expressing an AAV vector genome along with MV Rep proteins Rep78 and Rep68 in the isolated host cell according to claim 1 .
10. An isolated host cell, comprising a nucleic acid encoding Adeno-associated virus (AAV) Rep proteins Rep78 and Rep68 operably linked to an inducible promoter, wherein the internal AAV promoter p19 comprises a substitution at position 818 from a guanine to an adenine wherein the position 818 is numbered according to a complete AAV2 genome of GenBank Accession number AF043303, whereby the internal AAV promoter p19 is inactivated and the functionality of said Rep78 and said Rep68 proteins is maintained, wherein the nucleic acid also has at least 70% sequence identity to SEQ ID NO: 45.