Neurophage: orthogonal molecular engineering of the M13 phage for targeted delivery to the central nervous system

The blood-brain barrier (BBB) poses a formidable obstacle to central nervous system (CNS) drug delivery, blocking over 98% of potential neurotherapeutics. This thesis details the design, engineering, and validation of a versatile nanoplatform based on the M13 bacteriophage, developed to enable cell-specific targeted CNS delivery. Initially, the wild-typeM13 phage was shown to possess an intrinsic capacity to cross in vitro 2D and 3DBBB models via a non-disruptive transcytosis, maintaining structural and functional integrity. To enhance this ability, phage display was employed to engineer M13 variants presenting BBB-interacting peptides on the major coat protein (pVIII), resulting in significantly increased translocation compared to the wild-type virion. To further validate the platform, an orthogonally engineered phage was produced developed to simultaneously display a barrier-crossing moiety on pVIII and an anti-ALFA Tag nanobody on the minor coat protein pIII. This orthogonal nanovector efficiently crossed the 2D BBB model, retaining its specific targeting of primary neurons transfected with an ALFAtag-CD4 receptor, thereby validating the platform's ability to overcome sequential biological barriers. This work highlights the platform as promising tool to address one a major challenge in modern medicine: the efficient delivery of therapeutic and diagnostic molecules into the CNS. Its versatility supports multiple applications, ranging from complex neurodegenerative disorders, like Parkinson’s disease, to highly aggressive CNS malignancies like glioblastoma, where innovative and targeted treatment strategies are urgently needed. Thus, the therapeutic potential of this technology was explored in a glioblastoma model. An M13 phage targeting the EGFR-vIII oncomarker and functionalized with the photosensitizer Rose Bengal was applied for Photodynamic Therapy. Upon light irradiation, this system induced potent and specific apoptotic killing of target cancer cells, highlighting its potential to induce an immunogenic response. Collectively, these findings establish the M13 bacteriophage as a modular, effective, and rationally designable scaffold for next-generation, non-invasive, and targeted CNS therapies.