Main
Therapeutic interventions involving genome editing require the safe and effective delivery of molecules into target cell nuclei1,2,3. Although such capability would transform both clinical and research applications, current non-viral delivery is limited to cells treated ex vivo4,5,6, tissues targeted by local administration7,8 or the liver because of its natural propensity for molecular uptake8,9. Recent lipid nanoparticle formulations have been described with tropism for non-hepatic cells or organs10,11, but expansion of in vivo genome editing applications will probably require multiple approaches for molecular delivery to specific cells or organs inside the body following systemic administration.
Retargeting the tropism of viruses or viral vectors is an established delivery strategy involving the surface display of a cell-selective targeting molecule alongside a viral glycoprotein required for cell entry by fusion at the plasma membrane or in the low-pH environment of the endosome12,13,14,15. Recent progress leverages a mutant form of the vesicular stomatitis virus glycoprotein (VSVG), VSVGmut, that maintains endosomal fusion activity but lacks native low-density lipoprotein receptor binding affinity16,17,18. Pairing VSVGmut with cell-specific targeting molecules can redirect lentiviral transgene delivery and has enabled high-throughput screening of T cell and B cell receptor libraries to study receptor–antigen interactions19,20.
Particles cloaked in cellular membrane fragments—such as retrovirus-like particles (VLPs), extracellular vesicles and biomimetic nanoparticles—are gaining in popularity for the delivery of molecular cargo. For this class of EDVs, bioengineering is required to achieve molecular cargo packaging and control of targeting and fusogenic activity. Here, we show that human cell-specific genome editing can be achieved both ex vivo and in vivo by pairing the display of VSVGmut with antibody-derived single-chain variable fragments (scFvs) on EDVs that package Cas9 ribonucleoprotein (RNP) complexes (Cas9-EDVs). EDVs described in this paper leverage retroviral VLP assembly for the transient delivery of Cas9 RNPs8,21,22,23,24,25,26,27. We find that Cas9-EDVs achieve targeted genome editing within in vivo-generated chimeric antigen receptor (CAR) T cells in mice with a humanized immune system, with no off-target delivery to liver hepatocytes. These data show that EDVs are a programmable platform for delivering molecular cargo to specific cell types for complex genome engineering—both gene delivery and targeted gene disruption—inside the body.
Results
Receptor-mediated delivery and genome editing with Cas9-EDVs
A major challenge for in vivo delivery of editing enzymes is the lack of vehicles capable of targeting specific cell types. VLPs can package Cas9 RNP complexes produced by over-expressing Cas9 fused to the carboxy-terminal end of the viral Gag polyprotein during VLP production, but cell-selective VLP targeting has relied on cell infection strategies evolved by enveloped viruses22. To test whether VLPs could be reformulated as programmable EDVs, we first cloned a CD19 targeting antibody as an scFv fused to the stalk and transmembrane domain of CD8a, a strategy commonly used in CAR architecture28 (Fig. 1a and Supplementary Fig. 1a,b). Given that Cas9-VLPs bud from the plasma membrane of transfected producer cells, we reasoned that co-expression of the scFv fusion and VSVGmut together with lentiviral components that are necessary for Cas9 RNP encapsulation would generate Cas9-EDVs possessing both receptor specificity and endosomal escape capability, respectively.
Fig. 1: Cell-specific genome editing with antibody-targeted Cas9-EDVs.
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 0.05). For all plots, black lines indicate the median of the data set. LOD, limit of detection as defined by the average modified reads from lentiviral-treated samples.
T cell-targeting Cas9-EDVs containing the CAR transgene (n = 4) or T cell-targeting lentivirus containing the CAR transgene (n = 3) were systemically administered and in vivo cell engineering was assessed 10 days post treatment (Fig. 4b). CAR-transduced T cells were observed in all mice in which human cells successfully engrafted, as detected by mCherry expression (Fig. 4c,d and Supplementary Fig. 6a–c). In the two Cas9-EDV-treated mice that successfully engrafted with human T cells (n = 2 out of 4), we observed 1.67% and 1.51% modified alleles in the CAR-transduced T cells, compared to 0.04% and 0.04% in the CAR− T cells isolated from the same mice (Fig. 4e,f). As expected, no modified alleles were observed in cells isolated from mice treated with the T cell-targeted lentivirus. We repeated this experiment with mice humanized with PBMCs from a different donor and with more mice per treatment group, and we again observed CAR T cells generated in vivo in eight out of eight mice treated with T cell-targeted Cas9-EDVs and eight out of eight mice treated with T cell-targeted lentivirus (~0.5% vs ~5% CAR+ T cells, respectively) (Fig. 4g and Supplementary Fig. 6d–f). Again, we observed genome editing only in mice (n = 4 out of 8) treated with Cas9-EDVs, with higher levels of genome editing in CAR-transduced T cells than in CAR− T cells (Fig. 4h, i). Treatment with the T cell-targeted Cas9-EDV and lentivirus was well tolerated, with no weight loss observed (Supplementary Fig. 6g). Although mCherry+ F4/80+ Kupffer cells/macrophages were observed, no mCherry+ β-catenin-expressing hepatocytes were detected in the liver (Supplementary Fig. 7a–d). Together, these results indicate that antibody-based targeting of Cas9-EDVs is a strategy that maintains cell-selective and tissue-specific delivery of transgenes and genome editors in vivo.
The primary objective of our humanized mouse experiments was to assess Cas9-EDVs for their ability to mediate cell-targeted genome editing and transgene delivery in vivo. Given that human CD19+ B cells, in addition to T cells, engrafted in the second mouse cohort, we additionally assessed in vivo CAR T cell killing activity. Variable levels of CD19+ B cells were observed in Cas9-EDV-treated mice, and no CD19+ B cells were detected in mice treated with antibody-targeted lentivirus, demonstrating in vivo CAR T cell-mediated cytotoxicity (Fig. 5a and Supplementary Fig. 8a). This analysis suggests a model in which antibody-derived targeting molecules can direct molecular cargo to specific cells in vivo to successfully reprogram cell activity (Fig. 5b). Diverse T cell clonotypes were observed for CAR-transduced T cells isolated from mice in both groups (Fig. 5c), suggesting that multiple cells were engineered in vivo and did not arise solely through expansion of a single engineered cell. Given that clonotype diversity correlated with the number of CAR T cells analyzed (Supplementary Fig. 8b), the clearance of B cells in the lentiviral group was probably attributable to a higher number of CAR T cells generated during the initial in vivo transduction. Taken together, these findings offer an approach for generating genome-engineered cells with complex edits that could prove valuable for a wide range of clinical applications in the future.
Fig. 5: Functional dynamics of cellular engineering in vivo.
a, Depletion of CD19+ B cells is observed post administration of T cell-targeted lentivirus (experiment 2). Human CD45+ cells were isolated from PBMC-humanized spleens 10 days post systemic administration of T cell-targeted Cas9-EDV (n = 8 animals), lentivirus (n = 8 animals) or PBS (n = 4 animals), and the percentage of CD19-expressing cells was assessed by flow cytometry. P values calculated by means of Dunnett’s multiple comparison test after ordinary one-way ANOVA. **P
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