In vivo self-assembly and targeted delivery of EGFR siRNA-encapsulating small extracellular vesicles to EGFR-positive cancer cells for NSCLC therapy

We designed a synthetic biology circuit encoding both an EGFR-targeting peptide (GE11) and an EGFR siRNA under the control of the cytomegalovirus (CMV) promoter. After uptake by mouse liver cells via intravenous injection, this genetic circuit can reprogram the liver cells to produce and self-assemble EGFR siRNAs into secretory small extracellular vesicles (sEVs) tagged with the GE11 peptide, after which the siRNA-encapsulating sEVs are further transported via the blood circulation and guided by the GE11 peptide to EGFR-positive tumour cells for NSCLC therapy. RNA sequencing-based transcriptomic profiling confirmed the safety and low off-target activity of in vivo self-assembled EGFR siRNAs. Overall design: EGFR DEL19 transgenic mice were administered doxycycline for 25 days and analysed by micro-CT to ensure spontaneous tumour formation in the lungs. Mice were then intravenously injected with the CMV-scrR, CMV-siRE or CMV-GE11-siRE circuit (10 mg/kg) every 2 days for a total of 7 injections. After treatment, mice were sacrificed, and normal tissues and tumours were harvested to determine the global transcriptome alterations by RNA sequencing.