Retroviral gene transfer is the preferred method for stable, long-term integration of genetic material into cellular genomes, commonly used to generate chimeric antigen receptor (CAR)-T cells designed to target tumor antigens. However, the efficiency of retroviral gene transfer is often limited by low transduction rates due to low vector titers and electrostatic repulsion between viral particles and cellular membranes. To overcome these limitations, peptide nanofibrils (PNFs) can be applied as transduction enhancers. Among these, PNFs derived from the 12-mer peptide EF-C are well-investigated and commercially available. EF-C PNFs enhance transduction by forming EF-C PNFs/virus complexes that overcome electrostatic repulsion through their polycationic surface and interaction with cellular protrusions. However, the safe application of PNFs as transduction enhancers in gene therapeutic applications requires a fundamental understanding of their transduction-enhancing mechanisms, uptake, and degradation. In this study, we demonstrate that EF-C PNFs induce plasma membrane invaginations, increasing the membrane surface for viral attachment and reducing the distance to the nuclear membrane, thereby facilitating viral entry and transport to the nucleus. Furthermore, we identified macropinocytosis as the main entry pathway for EF-C PNFs and their subsequent degradation by lysosomal peptidases. The lysosomal degradation of EF-C PNFs prevents their accumulation as amyloid deposits, mitigating potential side effects and supporting their safe use in clinical applications.

Central Facility for Electron Microscopy Ulm University Ulm 89081 Germany

Central Facility for Electron Microscopy Ulm University Ulm 89081 Germany; Department of Ecosystem Biology Faculty of Science University of South Bohemia České Budějovice 37005 Czech Republic

Institute of Molecular Virology Ulm University Medical Center Ulm 89081 Germany

Institute of Molecular Virology Ulm University Medical Center Ulm 89081 Germany; Institute of Virology Philipps University Marburg Marburg 35043 Germany

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