Efficient and targeted delivery of genetic cargo remains a major barrier for gene and cell therapies, particularly in primary human T cells that are intrinsically resistant to viral transduction. Adeno-associated viruses (AAVs) are widely used vectors but are limited by immune neutralization, inefficient transduction of certain cell types, and lack of targeting specificity. Here, we present NEO-AAV, an engineered three-component fusion protein (PH-ALG2-PKD12) that is designed to recruit assembled AAV capsids into endogenous extracellular vesicles (EVs). The design couples PI(4,5)P2-directed membrane targeting, multivalent PKD12 (AAV receptor-derived PKD1-PKD2 tandem domain)-capsid clustering, and ALG2-mediated endosomal sorting complexes required for transport (ESCRT) machinery recruitment to redirect AAV biogenesis toward vesicle-mediated release, yielding EV-enveloped viral particles with improved vesicle loading compared with passively released EV-AAV controls. In neutralization assays, NEO-AAV displayed enhanced resistance to a monoclonal anti-AAV6 neutralizing antibody (ADK6), maintaining transduction activity across all tested antibody concentrations that fully neutralized naked AAV6 and exceeded the shielding capacity of passively formed EV-AAV6. By surface-displaying a CD7/CD3/CD28 tri-chimera, NEO-AAV6 concurrently activated and transduced primary human T cells in a single step, reaching ~40% eGFP+ cells among CD7+ lymphocytes, without requiring a separate bead-based pre-activation step. As a representative application, we demonstrate that NEO-AAV supports the generation of functional CAR-T cells from primary human peripheral blood mononuclear cells (PBMCs), with resulting CAR-T cells exhibiting antigen-specific IFN-gamma release and cytotoxicity against CD19+ Nalm6 target cells. Consistent with episomal AAV expression, CAR expression peaked near day 5 and declined by day 15, framing NEO-AAV as an activation-coupled delivery module rather than a stand-alone durable engineering solution. Together, these findings present NEO-AAV as a proof-of-concept, engineered secretion route for EV-enveloped AAV that improves immune shielding and enables single-step, activation-coupled transduction of CD7-expressing lymphocytes in primary human PBMCs, providing a building block for immune cell engineering workflows with potential applicability to in vivo settings.
Lei, Z., Xie, S., Yang, Q., Jansen, L. V., Yao, B., Yang, G., Qu, K., Vader, P., Snijders Blok, C., Jager, S. C. A., Boink, G. J. J., Schiffelers, R. M., Doevendans, P. A. F., Xiao, J., Sluijter, J. P. G.
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