RNA interference (RNAi) has emerged as a powerful strategy for selectively silencing disease-related genes by targeting mRNA before translation. Despite its promise, clinical translation of RNAi therapy has been hindered by the persistent challenge of efficient in vivo delivery. The field has evolved through two major phases: first, direct injection of synthetic siRNAs or chemically modified variants, and second, delivery via artificial vehicles such as lipid nanoparticles or conjugated ligands like GalNAc. While these approaches have led to FDA-approved drugs such as Patisiran and Givosiran, they still face significant limitations—particularly in delivering siRNAs beyond the liver, due to poor tissue specificity, immunogenicity, and instability in circulation.
To overcome these barriers, we developed a novel, proof-of-principle strategy that reprograms the host liver using programmable genetic circuits to produce and self-assemble siRNAs into exosomes that are naturally secreted into the bloodstream. This approach leverages the body’s own extracellular vesicle system, enabling systemic and targeted delivery without relying on artificial carriers. By integrating modular genetic components, we engineered cells to autonomously generate siRNAs, load them into exosomes, and release them into circulation—effectively turning the liver into a living bioreactor for therapeutic RNA production.
We designed a hierarchical genetic circuit architecture centered on a promoter-driven siRNA expression cassette. Using the CMV promoter to drive transcription of an optimized pre-miR-155 backbone containing an EGFR-targeting siRNA sequence, we achieved high guide strand expression with minimal passenger strand contamination—a critical improvement over shRNA-based systems.Nrf2 Antibody Data Sheet In vitro testing confirmed that HEK293T and Hepa 1-6 cells transfected with this construct efficiently produced functional siRNAs that were successfully packaged into exosomes, as validated by nanoparticle tracking analysis, electron microscopy, and enrichment of exosomal markers (CD63, TSG101, CD9). Furthermore, these exosomes effectively delivered siRNAs to recipient cells, resulting in dose-dependent knockdown of EGFR expression in mouse lung carcinoma cells.
To enable tissue-specific delivery, we incorporated a composable guidance module by fusing a brain-targeting RVG peptide to Lamp2b, a canonical exosome membrane protein. When introduced into the circuit, this modification directed exosomes to cross the blood-brain barrier and deliver siRNAs to neural tissues. Experiments in glioblastoma-bearing mice demonstrated that intravenously injected RVG-tagged exosomes significantly reduced tumor growth, suppressed both EGFR and TNC expression, and prolonged survival—highlighting the potential of this platform for treating CNS diseases.
Moreover, we extended the system to target multiple genes simultaneously. A dual-siRNA circuit co-expressing EGFR and TNC siRNAs was shown to achieve coordinated silencing in U87MG glioblastoma cells, confirming the scalability of our design. Importantly, all modifications preserved exosome integrity and functionality, indicating that the system is robust and modular.
In vivo studies revealed that after tail vein injection of the core circuit, siRNA precursors accumulated rapidly in the liver and were processed into mature forms within hours. Functional siRNAs were detected in plasma exosomes, followed by widespread distribution across multiple organs—including lung, pancreas, spleen, and kidney—with peak levels occurring at 9–12 hours post-injection. Notably, even low plasma concentrations of self-assembled siRNAs achieved potent gene silencing, reaching approximately 2400 copies per cell in the liver and 120 copies per cell in the lung—levels sufficient to trigger effective RNAi responses.
Therapeutic validation in mouse models underscored the strategy’s clinical relevance. In orthotopic lung cancer models driven by EGFR, treatment with the CMV-siRE circuit dramatically reduced tumor burden, reversed histopathological changes, and significantly improved survival.Phospho-PDPK1(Ser241) Antibody Epigenetic Reader Domain Similarly, in KRAS-driven spontaneous lung adenocarcinoma models, the CMV-siRK circuit led to marked reductions in tumor number and volume, accompanied by decreased phosphorylation of AKT and ERK—key downstream effectors of the KRAS pathway.PMID:34293804
In obesity models, we applied the same principle to target hypothalamic PTP1B, a key regulator of leptin and insulin signaling. Intravenous delivery of the CMV-RVG-siRP circuit resulted in significant weight loss, reduced adiposity, enhanced energy expenditure, and improved glucose homeostasis—effects attributed to restored central sensitivity to both leptin and insulin. These findings suggest that our platform can be adapted to treat complex metabolic disorders by targeting multiple pathways simultaneously.
Critically, the system exhibited excellent biocompatibility. Repeated administration caused no significant hepatotoxicity, renal damage, immune activation, or alterations in peripheral immune cell counts. The absence of inflammatory cytokine elevation further supports the safety profile of this endogenous delivery mechanism.
This work represents a paradigm shift in RNAi therapeutics. Rather than relying on pre-assembled synthetic complexes, our strategy enables *in vivo* self-assembly of siRNAs into natural exosomes, ensuring higher bioactivity, better stability, and enhanced targeting capability. It combines the precision of synthetic biology with the biocompatibility of endogenous transport mechanisms, offering a versatile, scalable, and safe platform for treating a wide range of diseases—from cancers to neurological and metabolic disorders.
Ultimately, this approach transcends the limitations of conventional delivery systems, paving the way for a new generation of RNAi therapies that are not only more effective but also inherently safer and more adaptable. By harnessing the body’s innate machinery, we bring RNAi closer to fulfilling its full therapeutic potential.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com