In vivo human T cell engineering with enveloped delivery vehicles

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Researchers at the University of California, Berkeley show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

Cell-specific genome editing with antibody-targeted Cas9-EDVs

Fig. 1

a, Schematic scFv targeting molecules (blue) and VSVGmut (orange) on the exterior surface of a Cas9-EDV. Cas9-EDVs package pre-formed Cas9-sgRNA complexes to avoid genetically encoding genome editors within a viral genome. b, Experimental outline and schematic of the lentiviral vector used for engineering HEK293T EGFP cells that express heterologous ligands on the plasma membrane (for example, CD19). To promote cellular engineering by single lentiviral integration events, engineered cell mixtures were generated through low multiplicity of infection to achieve <25% EGFP+ cells. Engineered cell mixtures were challenged with B2M-targeting Cas9-EDVs to test targeting molecule activity. ce, Assessment of antibody-targeted Cas9-EDV activity. HEK293T and CD19 EGFP HEK293T cells were mixed at an approximate ratio of 3:1 and treated with B2M-targeting Cas9-EDVs displaying various targeting molecule pseudotypes. Cas9-EDVs were concentrated 10× and cells were treated with 50 μl Cas9-EDVs (c,e) or in a dilution curve (d). Analysis was performed 7 days post treatment to assess B2M knockout in EGFP+ (on-target) and EGFP (bystander) cells by flow cytometry (c,d) and amplicon sequencing (e). n = 3 technical replicates were used in all experiments except for the 100 μl dose of CD19-scFv in d (n = 2). Individual replicate values and four-parameter non-linear regression curves are plotted in d. Error bars in e, s.e.m.

Hamilton JR, Chen E, Perez BS, Sandoval Espinoza CR, Kang MH, Trinidad M, Ngo W, Doudna JA. (2024) In vivo human T cell engineering with enveloped delivery vehicles. Nat Biotechnol [Epub ahead of print]. [article]

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