In vivo human T cell engineering with enveloped delivery vehicles

Abstract

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. Here, we 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.

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 <25% EGFP⁺ cells. Engineered cell mixtures were challenged with B2M-targeting Cas9-EDVs to test targeting molecule activity. c–e, 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. Source data

Fig. 2. Optimization of Cas9-EDVs for enhanced genome editing activity in primary human cells.

a, Genome editing activity comparison of CD19 antibody-targeted Cas9-EDV variants packaging B2M-targeted Cas9 RNPs. Expression of B2M protein was assessed by flow cytometry 7 days post treatment in CD19-expressing target cells. b, Diagram of the optimized Gag-Cas9 and Gag-pol Cas9-EDV production plasmids; features updated from a previous study are highlighted in teal. c–e, Genome editing activity of optimized VSVG-pseudotyped Cas9-EDVs in primary human CD34⁺ cells (c) and activated (d) and resting primary human T cells (e). B2M or TRAC genome editing was assessed by amplicon sequencing 7 days post treatment; n = 3 technical replicates were assessed for all conditions except for the untreated resting human T cells (n = 2). f, Schematic of potential intra-particle Cas9-EDV configurations for packaged Cas9 RNPs following proteolytic maturation. g, Schematic of the compound GS-CA1 inhibiting either the nuclear import and/or uncoating of an HIV-1 capsid. h, An mNeonGreen lentiviral vector was used to transduce HEK293T cells at the indicated multiplicity of infection (MOI) in the presence of GS-CA1 or DMSO. The percent of mNeonGreen-positive cells was assessed by flow cytometry 3 days post treatment. TU, transducing units. i, B2M-targeting Cas9-EDVs, pre-titered such that the highest treatment dose would result in approximately 50% of cells B2M⁻, were used to transduce HEK293T cells in the presence of GS-CA1 or DMSO. B2M expression was assessed by flow cytometry 3 days post treatment. Error bars, s.e.m. Unless otherwise noted, n = 3 technical replicates were used in all experiments; four-parameter non-linear regression curves are plotted in a, h and i.

Fig. 3. Multiplexed antibody targeting and editing of primary human T cells.

a,b, Treating resting human T cells with Cas9-EDVs co-displaying CD3 and CD28 scFvs results in cellular activation (a) and proliferation (b) as measured by flow cytometry detection of CD25 3 days post treatment and fold expansion relative to the untreated T cell count, respectively. CD25 expression and cellular proliferation was observed for CD3/CD28 scFv Cas9-EDVs, regardless of whether they packaged Cas9 RNPs targeting PDCD1 or a non-targeting control. c, Genome editing 3 days post treatment, as detected by amplicon next-generation sequencing. For a–c, Cas9-EDVs were concentrated 62× and 50 μl was used to treat 30,000 resting T cells. CD3 scFv-1 and CD28 scFv-2 were tested. d, Screening the mono-display of additional CD3 scFv targeting molecules for B2M-targeted Cas9-EDVs on the Jurkat T cell line. B2M expression was assessed by flow cytometry 3 days post treatment. Cas9-EDVs were concentrated 15× and 50 μl was used to treat 30,000 Jurkat cells. e, Testing a panel of T cell-targeted, B2M-targeting Cas9-EDVs, displaying single or multiplexed scFv targeting molecules. Activated primary human T cells were treated with 1.38 × 10⁸ Cas9-EDVs, displaying one or a combination of CD3 scFv-3, CD4 scFv-2, CD28 scFv-2 and a control scFv, and were assessed for B2M expression in CD4⁺ and CD8⁺ T cells by flow cytometry 6 days post treatment. Error bars, s.e.m.; n = 3 technical replicates were used in all experiments.

Fig. 4. Programmable human cell delivery generates gene-edited CAR T cells in vivo.

a, Summary of T cell-targeted Cas9-EDVs and lentivirus tested in PBMC-humanized mice. Both particles display multiplexed scFvs (CD3 scFv-3, CD4 scFv-1 and CD28 scFv-2). The Cas9-EDV vector co-packages a lentiviral-encoded CAR-2A-mCherry transgene and Cas9 RNP complexes to disrupt the TRAC gene; the lentivirus encodes the CAR-2A-mCherry transgene. b, Experimental schematic for testing T cell-targeted Cas9-EDVs and lentivirus in PBMC-humanized mice by intravenous (I.V.) retro-orbital injections. c, Representative flow cytometry plots demonstrating that CAR-expressing human T cells are detectable in the spleens of PBMC-humanized mice 10 days post administration of 1.5 × 10⁹ Cas9-EDV (n = 2 animals) or lentivirus (n = 3 animals) but not in mice administered PBS (n = 3), quantified in d. e,f, Gene editing is observed in CAR⁻ and CAR⁺ human T cells isolated from mice treated with T cell-targeted Cas9-EDVs (n = 2 animals) and T cell-targeted lentivirus (n = 3 animals). One CAR⁺ lentivirus sample was excluded in f because of failing sequencing. g, CAR-expressing human T cells are detectable in the spleens of PBMC-humanized mice 10 days post administration of 6.2 × 10⁸ Cas9-EDV (n = 8 animals) or lentivirus (n = 8 animals) but not in mice administered PBS (n = 4 animals). P values calculated by means of Dunnett’s multiple comparison test after Brown–Forsythe and Welsh one-way ANOVA. h,i, Genome editing is observed in CAR⁻ and CAR⁺ human T cells isolated from mice treated with T cell-targeted Cas9-EDVs (n = 8 animals per group). Significance calculated by two-sided unpaired t-test. Comparison in i is not significant (P > 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.

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 < 0.01. Black lines indicate the median of the data set. b, Model for the in vivo generation of CAR T cells, with or without simultaneous genome editing. Schematic made with Biorender and is not to scale. c, T cell receptor clonotypes of CAR-transduced T cells isolated from humanized mice treated with T cell-targeted Cas9-EDV (mouse numbers 2, 4, 6) or lentivirus (mouse numbers 13, 14, 17) from experiment 2. ‘n’ indicates the unique number of clonotypes detected.