Engineering Bacteriophage for genetic and regenerative therapies

There is need for improved gene delivery vectors which are efficient, safe, reproducible and cost-effective to allow transition into clinical application for regenerative medicine and tissue engineering purposes. The success of vector delivery techniques is dependent on the cell type of interest, the size of the transgene and whether transient or stable gene expression is required (Donnelly, et al., 2015) (Kim & Eberwine, 2010). Many techniques have been developed to deliver foreign DNA to modify target cell gene expression, including the adaptation of animal viruses to carry transgenes of interest, creation of DNA conjugates and physical methods (Larocca, 2002); (Mintzer & Simanek, 2008). However, each of these techniques is accompanied with significant disadvantages which limit their in vivo applications.

We demonstrated a protein based delivery system of functional cargoes. The technique, known as GAG-binding enhanced transduction (GET) includes the use of the protein transduction domain (cell penetrating peptide) polyarginine (8R) and the 21-residue heparin-binding domain from heparin-binding epidermal growth factor known as P21 (Dixon et al 2016).

Phage require modification to enable them to overcome mammalian cellular barriers. Donnelly et al, (2015) showed genetic modification of the M13 phage coat protein with an RGD ligand demonstrated higher efficiency of cell transduction when combined with traditional transfection reagents. Yet this was reliant on high volumes of transfection reagents which are both expensive and effect cell viability (Yamano, et al., 2010). To allow phage to successfully transduce mammalian cells, modifications to the surface coat are required. Cationic protein transduction domains (PTDs) have been derived from the HIV (HIV-1) TAT protein basic domain or engineered from poly-arginine or poly-lysine. Research has demonstrated their ability to deliver conjugated cargoes to modify cellular behaviour. It is thought that PTD delivery is mediated by endocytic pathways, such as lipid raft-dependent macropinocytosis (Gump, et al., 2010). This process is dependent on cytoskeletal rearrangements leading to encapsulation of contents within a macropinosome which then fuses with lysosomes to degrade contents (Lim & Gleeson, 2011). Therefore, to improve delivery, phage also need to express peptides that allow for endosomal escape.

This PhD will build upon preliminary work and the mini-project to show that genetic modification of M13 phage with GET peptides as a genetic fusion allows high efficiency transduction of mammalian cells. You will also demonstrate that phagemid containing a mammalian expression cassette can be encapsulated and delivered therapeutically, providing a basis for translation into in vivo gene delivery using phage as vector enhanced by GET peptides.