Biological gene delivery vehicles: beyond viral vectors

Abstract

Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications.

Figure 1

Examples of potential biological vehicles tolerated naturally in selected organs. Organisms and cell-derived particles are found naturally in certain organs as indicated, and are well-tolerated. These particles are promising delivery vehicles that can be exploited for gene therapy. AAV, adeno-associated virus.

Figure 2

Production of biological gene delivery vehicles. (A) Strains of bacteria with desirable properties are transformed with the plasmid cargo and amplified to generate GDVs. (B) The phagemid, a modified bacterial plasmid with phage sequences within, is used as the cargo and transformed into bacteria. The bacteria is then infected with a replication-defective helper phage that produces essential genes for the packaging of the phagemid vector into bacteriophage GDVs. (C) The virus surface proteins are produced in cell culture and purified as capsid monomers. The genetic cargo is then packaged into a virion as the monomers are transferred to a buffer that promotes assembly of the virion. (D) Erythrocytes are harvested from the patient and lysed to produce erythrocyte ghosts. The ghosts are then loaded, usually through osmotic pressure, with the genetic cargo before being reintroduced into the patient. (E) Patient-derived primary cells are first harvested and stimulated to produce exosomes, which are then purified, and loaded, likely by electroporation, with the genetic cargo before being reintroduced into the patient. GDV, gene delivery vehicle; MVB, multivesicular body.

Figure 3

Release of cargo intracellularly by delivery vehicles. (A) Bacteria can deliver genetic cargoes in two distinct fashion after endocytosis and endosomal release. First, short oligonucleotides and DNA plasmids can be released directly into the host cells through the lysis of the bacteria. Alternatively, intracellular bacteria can produce and excrete therapeutic RNAs and proteins. (B–D) Bacteriophage, VLPs and both types of liposomes are capable of delivering mRNAs, short oligonucleotides and DNA plasmids. (E) Viral vectors are typically only capable of delivering DNA or RNA vectors that ultimately end up in the nucleus as DNA templates for transcription of mRNAs. shRNA, short hairpin RNA; VLP, virus-like particle.