Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy.
The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT).
1] Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany [2] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany.
Sven
S
Hagen
1] Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany [2] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany.
Tobias
T
Baumann
1] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany [2] Institut für Biologie III, Albert-Ludwigs Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Hanna J
HJ
Wagner
1] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany [2] Institut für Biologie III, Albert-Ludwigs Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Volker
V
Morath
1] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany [2] Institut für Biologie III, Albert-Ludwigs Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Beate
B
Kaufmann
1] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany [2] Institut für Biologie III, Albert-Ludwigs Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Adrian
A
Fischer
1] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany [2] Institut für Biologie III, Albert-Ludwigs Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Stefan
S
Bergmann
1] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany [2] Institut für Biologie III, Albert-Ludwigs Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Patrick
P
Schindler
1] Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany [2] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany.
Katja M
KM
Arndt
1] Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany [2] BIOSS Centre for Biological Signalling Studies, Albert Ludwig University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany [3] Zelluläre und Molekulare Biotechnologie, Technische Fakultät, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
Kristian M
KM
Müller
D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D000229:Dependovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D009369:Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D011355:Prodrugs / Q000627:therapeutic use*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D011355:Prodrugs; D013937:Thymidine Kinase