Construction and utilization of a new generation of bacteriophage-based particles, or TPA, for guided systemic delivery of nucleic acids to tumors

Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood-brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques. This protocol describes the construction and production of transmorphic phage/adeno-associated virus particles for the targeted delivery of nucleic acid payloads, in vitro and in vivo.Transmorphic phage/adeno-associated virus particles ensure effective and safe delivery of nucleic acids (for example, luciferase, green fluorescent protein or cytokine genes) to solid tumors, thanks to their greater diffusion across the extracellular matrix and improved internalization and intracellular trafficking to the nucleus of target cells. This protocol covers the production and construction of new-generation transmorphic phage/adeno-associated virus vectors for the systemic delivery of nucleic acid payloads. In vitro and in vivo applications of transmorphic phage/adeno-associated virus particles are also discussed.