Steininger, Thomas and Öttl, Veronika and Franken, Linda E. and Frank, Cornelius and Ohland, Philip and Ferreiro, Miriam Lopez and Klostermann, Stefan and Fritsch, Johannes and Hirschauer, Evelyn and Sandmeir, Anna and Hilgenfeld, Luisa D. and Semmelmann, Florian and Duerr, Marie-Sofie and Konkel, Fabian and Pechmann, Gregor and Linder, Sabine and Haindl, Markus and Yazicioglu, Mustafa N. and Ringler, Philippe and Lauer, Matthias E. and Phichith, Denis and Seeber, Stefan and Fakhiri, Julia (2025) Improved Recombinant Adeno-Associated Viral Vector Production via Molecular Evolution of the Viral Rep Protein. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 26 (3): 1319. ISSN 1661-6596, 1422-0067
Full text not available from this repository. (Request a copy)Abstract
In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1-13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | IN-VITRO; VIRUS VECTORS; AAV VECTORS; GENE; TRANSDUCTION; EXPRESSION; SYSTEM; PURIFICATION; GENERATION; SEQUENCES; AAV; rAAV production; directed evolution; replication proteins; protein engineering |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 01 Apr 2026 11:33 |
| Last Modified: | 01 Apr 2026 11:33 |
| URI: | https://pred.uni-regensburg.de/id/eprint/67737 |
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