Molecular imaging of biological gene delivery vehicles for targeted cancer therapy: beyond viral vectors

Abstract

Cancer persists as one of the most devastating diseases in the world. Problems including metastasis and tumor resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of present clinical treatments. To overcome these limitations, cancer gene therapy has been developed over the last two decades for a broad spectrum of applications, from gene replacement and knockdown to vaccination, each with different requirements for gene delivery. So far, a number of genes and delivery vectors have been investigated, and significant progress has been made with several gene therapy modalities in clinical trials. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications. However, both have limitations and risks that restrict gene therapy applications, including the complexity of production, limited packaging capacity, and unfavorable immunological features. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents such as bacteria, bacteriophages, and bacteria-like particles. Recently, many molecular imaging techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been employed to assess vector-mediated gene expression in living subjects. In this review, molecular imaging techniques for monitoring biological gene delivery vehicles are described, and the specific use of these methods at different steps is illustrated. Linking molecular imaging to gene therapy will eventually help to develop novel gene delivery vehicles for preclinical study and support the development of future human applications.

Keywords: Cancer; Gene delivery vector; Gene therapy; Molecular imaging.

Fig. 1a–g

Production of biological gene delivery vehicles labeled with molecular imaging probes. a Strains of bacteria with desirable properties are transformed with the plasmid cargo. Imaging reporter genes such as the lux operon are transduced into the bacteria. b The bacteria are cotransformed with the plasmid encoding the therapeutic gene and the imaging reporter gene. c Docking of bacteria with functionalized multiple-sized nanoparticles through biotinylated antibodies and surface-antigen interactions (microbot). Streptavidin-coated nanoparticles can carry biotinylated cargo. d Minicells are derived from a minCDE-chromosomal deletion mutant of Salmonella enterica serovar Typhimurium (S. typhimurium). Target genes are incubated with minicells overnight. Bispecific antibody (BsAb) is used to target recombinant minicells to tumor cells. One arm of these antibodies recognizes the O-polysaccharide component of the minicell surface lipopolysaccharide and the other a tumor-preferential cell surface-receptor, such as the epidermal growth factor receptor (EGFR), which is overexpressed in several cancers. e The coat proteins of the bacteriophages can be engineered to incorporate targeting ligands. Phage nanoparticles with multiple peptides engineered for different functions can then be produced with phagemid technology to enhance gene delivery efficiency. f Retrovirus. g Adenovirus

Fig. 2a–g

Intracellular delivery of cargo by delivery vehicles. a Extracellular secretion of protein by bacteria, such as E. coli or S. typhimurium, which are defective in ppGpp synthesis. Proteins made by bacteria must be engineered with a leader secretory signal sequence (S) for extracellular (bacteria) secretion. For intracellular transport, bacterial proteins need to be fused to cell permeable peptide (CPP) sequences. b Intracellular delivery of genes/proteins by bacteria such as S. typhimurium, S. typhi or E. coli expressing the invasion of Y. pseudotuberculosis. The bacteria invade host cells and remain in the vacuole. There they die due to metabolic attenuation and release their expression plasmid or protein. By an unknown mechanism, the plasmids cross the vesicular membrane and reach the cell nucleus of the host cells where they are expressed. c Bacteria enter cells via induced phagocytosis. Bacterial toxin causes endosomal compartments to disintegrate. The therapeutic cargo then separates from the bacterium and is delivered to the nucleus. d Minicells carrying the target gene, such as siRNA, bind biomarkers on the surface of cancer cells via BsAb and enter the cell by endocytosis. After endocytosis, the minicells traverse the well-established early and late endosomal pathways, terminating in acidified organelles, the lysosomes, where they are degraded and release their cargo. e Bacteriophage nanoparticles carrying target molecules bind biomarkers on the surface of cancer cells via a targeting peptide and enter the cell by endocytosis. After endocytosis, the bacteriophages traverse the well-established early and late endosomal pathways, terminating in acidified organelles, the lysosomes, where they are degraded and release their cargo. f Retroviral gene transfection. g Adenoviral gene transfection

Fig. 3a, b

Specific gene expression in tumors by bacterial vectors. a A bacterial expression plasmid was constructed in which a Renilla luciferase variant (RLuc8) was placed under the control of a constitutive promoter (pLac-RLuc8). S. typhimurium defective in ppGpp synthesis (strain ΔppGpp) was transformed with the pLac-RLuc8 plasmid. The transformed bacteria were IV administered into immunocompetent BALB/c mice bearing CT-26. Due to early distribution of bacteria in the liver and spleen, bacterial bioluminescence was produced not only in the tumor, but also in the liver and spleen. b To verify that remote control of the bacterial gene expression is possible, a bacterial expression plasmid was constructed in which a Renilla luciferase variant (RLuc8) was placed under the control of the P_BAD promoter (pBAD-RLuc8). The mutant S. typhimurium ΔppGpp strain was electrotransformed with the pBAD-RLuc8 vector. The transformed bacteria were IV administered into immunocompetent BALB/c mice bearing CT-26. RLuc8 expression was induced with L-arabinose (60 mg) at 4 dpi. Bioluminescence was detected in the tumors after L-arabinose administration, but not in its absence. Bioluminescence was observed only in the tumor and not in the liver or spleen because the bacteria were cleared from these organs (liver, spleen) at 4 dpi