Targeted Organ-Specific Gene Delivery via Red Blood Cell Hitchhiking with Adeno-Associated Viruses

Introduction: Adeno-associated viruses (AAV) are leading gene delivery platforms due to their high transduction efficiency and safety. Efforts to enhance AAV-based therapeutics have focused on improving tissue tropism, transduction efficiency, and reducing immunogenicity, resulting in FDA-approved treatments for conditions like Duchenne muscular dystrophy and Leber congenital amaurosis. However, these improvements mainly involve virological aspects, such as serotype selection and capsid modifications, limiting AAV use to directly injectable tissues, like the retina and muscle, or those that naturally accumulate AAV after in vivo administration, such as the liver.

RBC hitchhiking (RH) presents an innovative approach to improving organ-specific AAV delivery and transduction. RH involves loading therapeutics onto RBC membranes, which then release drugs at target tissues after systemic administration. This method enhances initial AAV deposition through contact-mediated transfer from RBCs to the tissue endothelium, followed by shear-induced release of AAV, facilitating trans-endothelial migration and transduction. Previous studies have shown that RH enhances AAV delivery to the lungs and reduces off-target effects. This study expands on RH's potential by demonstrating its effectiveness in delivering AAV to the lungs via intravenous injection, achieving transgene expression beyond the endothelial layer. Combining RH with viral engineering holds promise for significantly improving the clinical efficacy of AAV-based gene therapies by ensuring targeted delivery and sustained exposure near endothelial cells.

Materials and

Methods: The RBC/AAV complex was formulated using a simple one-pot synthesis method by mixing RBC with AAV, tannic acid (TA), and FeCl3. For microfluidic chip studies, a single layer of the human endothelial cell line EA.hy926 was formed and treated with free AAV or RBC/AAV under flow or static conditions. For in vivo studies using mice, the formulations were intravenously injected via the tail vein. Viral titers in major organs were measured using qPCR.

Results, Conclusions, and Discussions:

Results: The RBC/AAV complex was formulated using a simple one-pot synthesis method by mixing RBC with AAV, tannic acid (TA), and FeCl3. The resulting RBC/AAV exhibited nano-scale clumps composed of AAV, TA, and Fe ions on the surface of the RBC membranes (Fig. 1A). In a microfluidic chamber, RBC/AAV significantly increased AAV deposition on endothelial cells compared to free AAV under flow conditions, facilitated by contact between RBCs and endothelial cells (Fig. 1B). The transduction rate was significantly higher when the flow was applied during RBC/AAV treatment compared to static conditions, indicating the role of shear-induced AAV release from the complex in enhancing transduction (Fig. 1C). In vivo, RH-mediated delivery resulted in a ~100-fold increase in lung accumulation of AAV (Fig. 1D).

Conclusions: Here we investigated how AAVs are displaced from the complex through shear and physical contact-based interaction with endothelial cells. The interactions between TA, AAV, and Fe lead to the complex formation on the RBC surface, followed by the release of AAV at target organs upon contact with the endothelium which is augmented by the shear-induced release. In target organs like the lung, the RBC/AAV complex encounters peripheral blood vessels, providing shear force and contact points that lead to AAV accumulation and transient increase in local AAV concentration near endothelial cells. This allows AAVs to permeate deeper into the tissue. In the lung, this permeation results in transgene expression past the endothelial layer into the lung interstitium.

Discussion: Efficient gene delivery to target organs is a critical challenge in successful gene therapy. However, this aspect is often overlooked in AAV-mediated gene therapy, where the focus has been on engineering viral vectors through capsid or gene cassette modifications, limiting AAV application. Here we achieved significant improvement in organ-specific delivery of AAV using RH. We further observed transduction beyond the endothelium, opening a huge potential for treating various genetic diseases that require transduction in parenchymal regions. We envision that optimizing RH technology with vector engineering and careful serotype selection will enhance AAV's tissue tropism and transcytosis ability, improving treatment efficacy for various diseases.

Acknowledgements (Optional):