Abstract

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.