Targeted Gene Delivery to Astrocytes Using Intelligent Phagemid-Assembled Gene Expression (iPhAGE) Technology for Neuronal Regeneration

Abstract

Neurodegenerative diseases, stroke, and brain injuries lead to progressive neuronal loss, with limited treatment options focused mainly on symptom management rather than regeneration. A major challenge in developing effective therapies is the lack of safe and efficient gene delivery systems capable of targeting cells in the central nervous system. This project addresses this gap by developing a novel gene therapy approach using the Intelligent Phagemid-Assembled Gene Expression (iPhAGE) platform, an innovative bacteriophage-based system designed to deliver reprogramming transcription factors to astrocytes. The iPhAGE system, derived from M13 miniphagemids, offers high transfection efficiency, large cargo capacity, low immunogenicity, and blood-brain barrier permeability, making it an ideal tool for targeted gene delivery. This research involves constructing plasmids carrying the NeuroD1 gene under astrocyte-specific and universal promoters, producing and characterizing miniphagemids, and evaluating their transfection efficiency in resting and activated A7 cells. Neuronal reprogramming is assessed through PSA-NCAM neuronal marker analysis, a marker associated with early neuronal differentiation, used to evaluate phenotypic changes following NeuroD1 delivery. Key methodologies include designing and validating precursor and helper plasmids, producing miniphagemids, and characterizing them using restriction digestion, MADLS, and transfection assays. The outcome of this project was the establishment and initial evaluation of the iPhAGE-based gene delivery framework for astrocyte-to-neuron reprogramming. This work assesses the main components of the iPhAGE system, including vector construction, miniphagemid production, and astrocyte reprogramming using neuronal markers, therefore establishing a groundwork for future optimization.