A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties

Abstract

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Keywords: BASP1; EV engineering; IL-12; PTGFRN; exosome engineering; exosomes; extracellular vesicles; sacffold; vaccine.

Graphical abstract

Figure 1

EV purification and characterization (A) Isolation of fractions F1–F4 from conditioned media. (B) Gradient image after 150,000 × g centrifugation highlighting F1–F4. (C) Density profile of blank iodixanol gradient after 150,000 × g centrifugation. (D) Representative transmission electron micrographs of F1–F4. (E) Cholesterol, DNA, protein, and particle concentrations in F1–F4. Data from 3 EV isolations are plotted as mean ± SD (F) Representative protein normalized SDS-PAGE of F1–F4, producer cell lysate (CL), and crude ultracentrifuged (UC) pellet with immunoblots for individual markers. Molecular weight markers for all immunoblots and SDS-PAGE gels are given in kDa. (G) Heatmap plotting quantitative peptide spectrum matches for proteins indicated. Proteins highly enriched in F1 (top) and F4 (bottom) are shown. Data are averaged from triplicate measurements from a representative EV isolation.

Figure 2

Characterization of scaffolds for EV engineering (A) EV membrane lipid bilayer showing different classes of scaffold proteins with luminal GFP fusions. (B) MFI of EV producer cells stably expressing GFP fusions to the indicated scaffold. Data from 3 biological replicates are plotted as mean ± SD (C) Average number of GFP molecules per EV determined by NTA and GFP ELISA. GFP standard curve used for quantitation shown in inset. Data from 3 biological replicates are plotted as mean ± SD. All statistical comparisons are given in Figure S2D. (D) Representative histograms from flow cytometry measurements of engineered EVs with GFP fused to the indicated scaffold. Data are normalized to the highest count within each sample.

Figure 3

Determination of scaffold requirements for EV engineering (A) EV membrane lipid bilayer showing GFP fusions to IgSF-EWI and MARCKS family members. Numbers for PTGFRN truncations indicate the first amino acid included from the PTGFRN protein sequence. “+++++,” polybasic region. (B) Average number of GFP molecules per EV determined by NTA and GFP ELISA. Data from 3 biological replicates are plotted as mean ± SD. B, BASP1; L, MARCKSL1; M, MARCKS. (C) EV normalized anti-GFP immunoblot of EVs with GFP fused to the indicated truncation. The 55-kDa membrane-bound cleaved product and putative ADAM10 cleavage site are shown. (D) EV normalized anti-GFP immunoblot following transient transfection of FL-GFP and Δ395-GFP into WT or ADAM10 knockout (−/−) producer cell lines with anti-ADAM10 immunoblot to confirm deletion. (E) EV-normalized SDS-PAGE and anti-GFP immunoblot of EVs with C-terminal GFP fusions to incremental truncations of BASP1 with amino acid positions and point mutations labeled. (F) EV-normalized anti-GFP immunoblot blot of EVs with C-terminal GFP fusions to single amino acid truncations of BASP1, with minimal sequence required for EV localization highlighted.

Figure 4

PTGFRN enables EV surface display of protein cargo (A) EV membrane highlighting the classes of proteins displayed by fusion to PTGFRN. (B) In vitro activity of EVs displaying IL-7. Mean values from 4 donors using 2 EV isolations were compared by 1-way ANOVA and a Tukey post hoc test. ∗p = 0.0108; Rec., recombinant IL-7. (C) In vitro activity of EVs displaying CD40L. Mean values from 4 donors using 2 EV isolations were compared by 1-way ANOVA and a Tukey post hoc test. ∗p = 0.0247; ∗∗∗∗p < 0.0001; Rec., recombinant CD40L ECD. (D) In vitro activity of EVs displaying αCD3 scFab. Mean values are shown from individual mouse spleens using 2 EV isolations. Rec., recombinant αCD3. (E) In vitro activity of EVs displaying IL-12. Mean values from 4 donors using 2 EV isolations were compared by 1-way ANOVA and a Tukey post hoc test (ng/mL) or an unpaired t test (p/mL). Rec., recombinant IL-12; ns, not significant. (F) In vivo activity of EVs displaying murine IL-12 (FL) compared to treatment with recombinant murine IL-12 (Rec.) at 100 and 200 ng doses. Percentages of reduction in tumor volume compared to PBS treatment are given. Independent EV isolations were used for each study and data were compared by 1-way ANOVA and a Tukey post hoc test. ∗p < 0.05; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001; ns, not significant. (G) Survival of EV-treated animals compared to recombinant murine IL-12. 100 ng, p = 0.0209; 200 ng, p = 0.0421 by Gehan-Breslow-Wilcoxon test.

Figure 5

BASP1 enables EV loading with protein cargo (A) EV membrane lipid bilayer showing proteins loaded with BASP1 along with vaccine formulations of OVA and adjuvant. Vaccination experiments in (B)–(D) were performed with independent EV isolations. (B) OVA and CDN combinations were administered i.n. as free compounds or associated with EVs. Lung and spleen effector memory T cells reactive to the dominant MHC class I epitope in OVA (SIINFEKL) were measured by flow cytometry (means ± SDs; n = 3 per group). ∗p < 0.0182; ∗∗∗∗p < 0.0001; for exoVACC compared to all groups by 1-way ANOVA. (C) OVA and CDN combinations were administered i.n., and ELISpot analysis on viable splenocytes is shown for reactivity against dominant CD4 and CD8 epitopes. Data are presented as background subtracted IFNγ spot-forming units (SFUs) per 100,000 splenocytes (means ± SDs; n = 5 per group). ∗p < 0.0168; ∗∗p < 0.0014; for exoVACC compared to all groups by 1-way ANOVA. (D) OVA-reactive splenic effector memory T cells measured following a single dose of exoVACC administered i.v., i.n., or s.c. alongside s.c. dose-matched free OVA formulated with AddaVax (mean ± SD; n = 5 per group). ∗p = 0.0162; ∗∗p = 0.0015; compared to OVA + AddaVax group by Welch’s 1-way ANOVA.