Genetically encoding multiple functionalities into extracellular vesicles for the targeted delivery of biologics to T cells

The genetic modification of T cells has advanced cellular immunotherapies, yet the delivery of biologics specifically to T cells remains challenging. Here we report a suite of methods for the genetic engineering of cells to produce extracellular vesicles (EVs)—which naturally encapsulate and transfer proteins and nucleic acids between cells—for the targeted delivery of biologics to T cells without the need for chemical modifications. Specifically, the engineered cells secreted EVs that actively loaded protein cargo via a protein tag and that displayed high-affinity T-cell-targeting domains and fusogenic glycoproteins. We validated the methods by engineering EVs that delivered Cas9–single-guide-RNA complexes to ablate the gene encoding the C-X-C chemokine co-receptor type 4 in primary human CD4⁺ T cells. The strategy is amenable to the targeted delivery of biologics to other cell types.