Serotype-specific tropism of adeno-associated viruses in dorsal meningeal lymphatic vessels via intra-cisterna magna delivery

Background Meningeal lymphatic vessels (mLVs) in the brain acts as the regulator that eliminates harmful macromolecules in post-cerebrospinal fluid (CSF) and facilitates immune cell migration to cervical lymph nodes. However, it still poses challenges on the investigation of separated mLVs biological functions because of its unique anatomical position on the dorsal skull. Studies have been revealed that the recombinant adeno-associated virus (rAAV)2/1 serotype can be recognized as delivery vehicles targeting mLVs, which further can be exploited to evaluate mLVs biological functions. Nevertheless, the characteristics for infection of rAAV2/1 needs more investigations. Methods In the present study, 8-week-old male C57BL/6 mice were selected, and rAAVs of different serotypes were injected either into the cisterna magna filled with CSF or via lateral ventricle injection, to compare the specificity of distinct rAAV serotypes for infecting mLVs. Results Through intracisternal magna delivery, rAAV2/1-CMV-EGFP exhibited selective mLVs targeting with sustained fluorescence intensity. Additionally, rAAV2/5-CMV-EGFP, rAAV2/8-CMV-EGFP, rAAV2/9-CMV-EGFP, rAAV2/BR1-CMV-EGFP, and rAAV2/PHPeB-CMV-EGFP also showed the same trend. Significantly, rAAV2/9-CMV-EGFP infectivity via the injection in medullary cisterna induces EGFP expression in mLVs that is dose-dependent. However, at higher viral doses (≥1×10 9 vg/mouse), expression reaches a plateau and maintains stability for 6 months, suggesting a saturation threshold of transduction efficiency. Conclusion This study revealed the characteristics of diverse rAAV serotypes in mLVs research, suggesting that rAAVs can be regarded as a powerful and safe gene therapy tool for unveiling the role of mLVs under neuroinflammatory conditions or central nervous system (CNS) disorders.