Evaluating M13-Based Targeted Gene Delivery for Alzheimer’s Disease Applications

Overview

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by progressive memory loss and cognitive decline. This eventually leads to impaired daily functioning, affecting behaviour, language, and motor skills, underscoring the critical need for innovative therapeutic strategies. Gene therapy offers significant promise, but its successful implementation in AD requires highly specific and efficient delivery systems capable of targeting affected neurons and overcoming biological barriers.

This project explored the potential of bacteriophage M13-based phagemid vectors as a novel targeted gene delivery system for Alzheimer's disease. Our approach focused on engineering phagemids to precisely deliver therapeutic genetic information to neuronal cells, addressing the molecular and cellular pathologies underlying AD. Early objectives included characterizing gene expression changes in Aβ-treated neurons to identify potential therapeutic targets, and subsequently selecting a specific neuropeptide for display on the phagemid's major capsid protein to facilitate neuronal targeting.

To achieve targeted delivery and controlled expression, specific modifications were made to the miniphagemids:

Targeting Ligand Display: The phagemids were engineered to display a selected neuropeptide, Angiotensin IV (AT4), on their major capsid protein:
- The receptor for AT4 is significantly expressed in brain regions vital for learning, memory, and cognitive processing. Crucially for neuronal delivery, this receptor is known to facilitate receptor-mediated transcytosis across the blood-brain barrier.
- This modification is crucial for targeted internalization. Engineering our M13 miniphagemids with AT4 allowed for targeted transport into the brain, ensuring that the miniphagemids are preferentially taken up by neuronal cells.
Therapeutic Gene Packaging: The phagemid vectors were designed to selectively package desired therapeutic genetic cargo:
- Zinc Import Protein 3 (ZIP3): ZIP3 has a critical role as a zinc transported within neurons. Zinc dysregulation is a contributing factor in neurodegenerative conditions, including AD.
- eGFP was also used, as a reporter
Cell-Type Specific Expression: Within the phagemid plasmids, a neuronal specific promoter (Synapsin 1) was incorporated.
- Synapsin 1 is expressed almost exclusively in neurons, making it an effective tool for restricting transgene expression
- Once delivered these promoters ensure that the therapeutic genetic material is transcribed in a cell-type specific manner, aiming to maximize therapeutic effect within neurons while minimizing off-target expression in other cell types.
- Elongation Factor 1α (EF1α) was also used as a control

There was also a focus on the production and comprehensive characterization of miniphagemids carrying the gene of interest, alongside evaluating the resulting gene expression changes in both neuronal and non-neuronal cell types.

Through this work, we have laid foundational groundwork for the application of M13-based phagemid vectors in targeted gene delivery for neurons. Our findings contribute to the understanding of how these phage-derived systems can be engineered for specific cellular internalization and expression. Continued development of these advanced vectors offers promising avenues for precise therapeutic interventions in Alzheimer's disease, paving the way for delivering genetic treatments directly to affected neural pathways.