Targeting Bacteriophage Delivery of Reprogramming Transcription Factor Genes to Astrocytes in the CNS

Overview

The filamentous bacteriophage M13 is a single-stranded DNA phage that co-exists with its Escherichia coli host after infection. M13 possesses several attractive characteristics for gene delivery including high titres of phage progeny, amenability to display of foreign peptides on the capsid, a simple, well-characterized genome, and flexible virion size. Filamentous bacteriophages also have a well-documented ability to cross the blood-brain barrier which is an exploitable trait that can be applied to targeted phage delivery to the central nervous system.

Astrocytes make up the majority of cells within the CNS and perform a multitude of roles including metabolic, structural, homeostatic, and neuroprotective tasks such as regulating the blood-brain barrier, promoting and maintaining synapses, and clearing excess cell waste. It has also been shown that astrocytes are able to differentiate into functional neurons. These unique abilities make them an ideal target for gene delivery and cell transformation as a potential treatment for a multitude of neurological diseases and disorders including stroke, epilepsy, traumatic brain injury, and Alzheimer’s disease.

We have previously demonstrated that linear covalently closed (LCC) minivectors (DNA ministrings or msDNA) offer a superior safety and transfection efficiency profile over their isogenic plasmid and even their minicircle counterparts. These LCC DNA minivectors are nonimmunogenic, redosable, and confer transfection efficiencies that rival some adenoviral vectors in several tissues. The vectors carry only a minimal expression cassette comprised of the eukaryotic promoter, gene of interest (GOI), intron and polyA sequence and nuclear translocation enhancing. The minivectors are also capable of a large capacity without dramatic impact on their cellular trafficking and nuclear translocation profile.

Intelligent Phagemid-Assembled Gene Expression (iPhAGE) System: We have recently developed a one-step continuous production platform for the generation of bacteriophage (phage) M13 mini phagemids engineered to carry LCC DNA minivectors in a targetable manner. Our system generates viable phagemid encapsulating the functional vector’s LCC minivector with theoretically limitless capacity and without any prokaryotic antibiotic resistance or origin of replication DNA.

The astrocyte-specific glial fibrillary acidic protein (GFAP) promoter will be used to drive expression of the reprogramming transcription factors neuroD1 or Sox-2 gene; each will be co-expressed with luciferase (luc) for imaging.

The goal of this project is to show that intravenous administration of M13 miniphagemids, engineered to carry LCC DNA vectors (iPhAGEs) encoding transcription factor neuroD1 or Sox 2 under the GFAP astrocyte-specific promoter will effectively cross the blood-brain barrier and specifically deliver a transcription factor for the reprogramming of astrocytes to neurons.