Multiscale red blood cell hitchhiking for targeted deep tissue gene delivery in lungs

Abstract

The clinical impact of gene therapies is constrained by poor delivery to target tissues beyond the liver after intravenous administration. Current molecular targeting strategies, such as capsid engineering or gene-carrier surface modification, have achieved only limited success due to their inability to overcome the hierarchical barriers from injection to deep tissue transduction. Here, we introduce a Multiscale Approach using RBC-mediated hitchhiking and Vascular Endothelium Leakage (MARVEL), which integrates red blood cell hitchhiking with VEGF-induced vascular permeabilization to enhance accumulation and penetration of cargoes. Using adeno-associated viruses (AAVs) as a model, MARVEL markedly increases AAV localization in the lungs, improves endothelial transcytosis, and enables gene expression in deeper tissue layers while maintaining a favorable safety profile. We further demonstrate that MARVEL can be adopted into an in situ hitchhiking approach, bypassing the need for ex vivo formulation. MARVEL provides a scalable strategy to address long-standing delivery challenges in gene therapy.

Fig. 1. Schematic description of RBC hitchhiking.

RBCs with surface-loaded AAV and VEGF are administered via intravenous or intra-arterial routes, after which they enter peripheral vessels in target organs (shown in the schematic: lung capillaries after intravenous injection). As the RBC complex squeezes through the capillaries, AAV and VEGF are dislodged through contact and shear forces. VEGF permeabilizes the endothelium by disrupting cellular junctions, allowing AAV infiltration and transduction in deeper tissues. TA: tannic acid. Created in BioRender. Park, C. (2025) https://BioRender.com/i75h519.

Fig. 2. Formulation process of RBC hitchhiking.

A Schematic description of MARVEL preparation through one-pot synthesis. B SEM images of i) bare RBC, ii) RBC loaded with AAV, and iii) TA/Fe/AAV complex. Representative images from two independent experiments. Scale bars (B) i) and ii)  = 1 µm, and iii) = 100 nm.

Fig. 3. RBC/AAV complex preparation and function characterizations in vitro and in vivo.

A The number of AAV6-CMV-Luc loaded per RBC (n = 3 independent biological replicates), B cumulative release in a mild shaking condition (n = 4 independent biological replicates), and (C) shear-induced release were measured by PCR (n = 3 independent biological replicates). D Confocal images of EA.hy926 cultured in a microfluidic chip were taken after a stream of AF647-labeled AAV6-CMV-Luc was given. AF647 and the nucleus are shown in red and blue, respectively. Representative images from two independent experiments. Scale bar = 50 µm. E AF647 signals were quantified from 3 independent planes on the confocal images. F In vitro transduction of EA.hy926 cells by RBC/AAV (AAV6-CMV-Luc) treated in a static or flow condition (n = 3 biological replicates for No Treatment, n = 7 for No Flow, n = 4 for Flow). Consolidated data from two independent experiments. G In vivo biodistribution of AAV6-CMV-Luc 1 h after intravenous injection measured with PCR, (H) percent distribution among the major organs, and (I) lung-to-liver ratio (n = 3 female C57BL/6 mice per group, 5–6 weeks of age). Statistical analysis was performed with unpaired, two-sided Student’s t-test (E, I), two-way ANOVA, followed by Sidak’s multiple comparisons test (G), and one-way ANOVA, followed by Tukey’s multiple comparisons test (F). All data are presented as mean ± SEM. Source data are provided as a Source Data file.

Fig. 4. MARVEL preparation and functional characterization in vitro and in vivo.

A VEGF loading on RBC was measured with ELISA (n = 3 independent biological replicates). B Confocal images of primary human brain endothelial cells after a 30-min incubation with 20 ng/mL VEGF with or without TA and Fe. No treatment (NC) served as the negative control, and a 24-h incubation with 100 ng/mL VEGF was used as the positive control (PC). Representative images are shown from n = 3 biological replicates per group. Blue: nucleus; green: VE-cadherin. Scale bar = 50 µm. VE-cadherin signals quantified from n = 7 planes (NC), n = 5 (free VEGF), and n = 6 (VEGF + TA+Fe and PC) from the confocal images. C VEGF amounts in the lungs 4 h after intravenous injection measured by ELISA (n = 4 female C57BL/6 mice per group, 5–6 weeks of age). The percentages of transduced (D) endothelial and (E) epithelial cells characterized by GFP+ signals in the lungs were measured with flow cytometry 30 days after intravenous injection with formulations containing AAV6-CMV-GFP and VEGF (n = 5 female C57BL/6 mice per group, 5–6 weeks of age). Statistical analysis was performed with (C, B, D, E) one-way ANOVA, followed by Tukey’s multiple comparisons. Data are presented as mean ± SEM. ns: not significant. Source data are provided as a Source Data file.

Fig. 5. In situ drug loading on RBC in whole blood and lung-specific targeting.

A Loading of fluorescent polystyrene beads (PS) with or without TA and Fe on RBC in whole blood in a static condition, measured with a plate reader (n = 3 independent biological replicates). B Binding of AAV6-CMV-Luc to RBCs in whole blood under static conditions confirmed by flow cytometry (n = 3 independent biological replicates). C Biodistribution of AAV6-CMV-Luc 1 h after intravenous injection of AAV alone or mixed with TA and Fe into the tail vein, and (D) the corresponding lung-to-liver ratio (n = 4 female C57BL/6 mice per group, 5–6 weeks of age). Percentages of transduced (E) endothelial and (F) epithelial cells characterized by GFP+ signals in the lungs measured by flow cytometry 30 days after intravenous injection of formulations containing AAV6-CMV-GFP (female C57BL/6 mice per group, 5–6 weeks of age; n = 5 for the no treatment group, and n = 7 for the treatment groups). Statistical analysis was performed with (B, D) unpaired, two-sided Student’s t-tests, (C) two-way ANOVA, followed by Sidak’s multiple comparisons test, or (E, F) one-way ANOVA, followed by Tukey’s multiple comparisons test. Data are presented as mean ± SEM. ns: not significant. Source data are provided as a Source Data file.