Background:
Alzheimer’s disease (AD) pathology involves amyloid-beta (Aβ) accumulation, triggering neuroinflammation and reactive astrogliosis. Microglia clear these aggregates via TAM receptor-mediated phagocytosis. Previously, Gas6-based variants were developed to bridge Aβ and TAM receptors for enhanced clearance. However, efficient whole-brain delivery and temporal control of expression remain critical bottlenecks. To overcome this, we proposed a novel non-invasive therapeutic approach: an engineered Gas6 (eGas6) variant delivered systemically via an Adeno-Associated Virus (AAV) system under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter. This design aims to achieve safe, disease-state-dependent conditional secretion aligned with astrogliosis progression across the entire brain.

Methods:
The eGas6 variant was molecularly designed for optimized structural stability and productivity. The gene was cloned into an AAV vector downstream of the GFAP promoter. To evaluate functional activity, in vitro phagocytosis assays were performed using immune cells treated with labeled Aβ aggregates, followed by flow cytometry (FACS) quantification. Crucially, to validate systemic in vivo feasibility, the AAV-GFAP-eGas6 platform was delivered via intravenous (IV) injection into C57BL/6 (B6) mice, enabling non-invasive targeting of the central nervous system. Expression, global distribution, and secretion profiles within the brain were assessed via biochemical and histological analyses.

Results:
In vitro functional assays confirmed that eGas6 successfully bound to TAM receptors and robustly accelerated the engulfment of Aβ species, showing a significant increase in the phagocytic index compared to control groups. Furthermore, in vivo evaluation in B6 mice demonstrated that a single systemic IV administration of the AAV-GFAP-eGas6 platform achieved successful blood-brain barrier (BBB) crossing and widespread, uniform protein expression throughout the whole brain. The engineered protein was effectively processed and secreted without inducing detectable adverse cell toxicity, confirming the baseline in vivo safety and robust non-invasive delivery efficiency of the vector system.

Conclusion:
In conclusion, we have successfully developed and validated a non-invasive, whole-brain AAV delivery platform for an engineered Gas6 (eGas6) utilizing a GFAP promoter. By confirming powerful in vitro phagocytic activity and widespread in vivo expression via systemic injection in wild-type mice, this study provides a solid proof-of-concept for a self-regulating, clinically viable immunotherapeutic strategy. These findings establish a rigorous foundation for subsequent efficacy evaluations in AD mouse models, highlighting the potential of eGas6 as a sophisticated platform technology for neurodegenerative diseases.