Nickl, P.; Barbiera, M.; Zini, J.; Nickl, T.; Ushiki, A.; Vaskovicova, M.; Neburkova, J.; Dolejs, V.; Simova, M.; Balounova, J.; Vyletal, P.; Zivna, M.; Kmoch, S.; Sumbalova-Koledova, Z.; Filipp, D.; Ballek, O.; Neiderlova, V.; Stepanek, O.; Ahituv, N.; Yliperttula, M.; Sedlacek, R.

Adeno-associated virus purification by density-gradient ultracentrifugation is labor-intensive and often results in substantial titer loss due to particle aggregation. Here, we present a scalable co-isolation strategy in which AAV is precipitated together with extracellular vesicles secreted by the producer cell line, completely bypassing density-gradient separation. The resulting AAV-EV preparations comprise free AAV, free EVs, and EV-associated AAV. Functionally, AAV-EV vectors (AAV2/1 serotype) support efficient ex vivo genome editing across multiple independent loci in mouse and rat zygotes, achieving a mean targeting efficiency of approximately 26%. Compared with gradient-purified AAV administered at matched doses, AAV-EV formulations yielded 2.34-fold higher embryo viability while maintaining equivalent transgene copy numbers. By leveraging EVs as a biological matrix, this approach enables ultracentrifugation-free AAV isolation without compromising vector functionality. Overall, AAV-EV represents an accessible and embryo-tolerant platform for rodent genome engineering that aligns with the principles of Replacement, Reduction, and Refinement (3R) principles.