Development of adenovirus capsid proteins for targeted therapeutic delivery

Abstract

The outer shell of the adenovirus capsid comprises three major types of protein (hexon, penton base and fiber) that perform the majority of functions facilitating the early stages of adenovirus infection. These stages include initial cell-surface binding followed by receptor-mediated endocytosis, endosomal penetration and cytosolic entry, and intracellular trafficking toward the nucleus. Numerous studies have shown that the penton base contributes to several of these steps and have supported the development of this protein into a delivery agent for therapeutic molecules. Studies revealing that the fiber and hexon bear unexpected properties of cell entry and/or nuclear homing have supported the development of these capsid proteins, as well into potential delivery vehicles. This review summarizes the findings to date of the protein-cell activities of these capsid proteins in the absence of the whole virus and their potential for therapeutic application with regard to the delivery of foreign molecules.

Figure 1. Representation of adenovirus capsid (inset) and the early stages of adenovirus infection

(A) Binding of the virus to the primary receptor, CAR, which initiates infection. This is immediately followed by (B) secondary binding of the virus to integrins, which triggers receptor-mediated endocytosis. CAR: Coxackievirus adenovirus receptor.

Figure 2. Representation of gene delivery complexes, 3PO and H2PO

Colored bars at the top of the figure represent the linear sequences of PBK10 and HerPBK10 from amino to carboxy termini (left to right). The structure of the assembled complex is speculated based on functional findings, including receptor-specific delivery and gene expression, DNA condensation (based on ethidium bromide exclusion), and DNA protection from serum nucleases. Data taken from [47,58].

Figure 3. 3PO gene transfer and comparison of cytotoxicity to Ad

(A) 3PO-mediated delivery of luciferase-expressing plasmid to HeLa plated in a 96-well dish (0.1 μg of DNA/well used). (B) GFP gene transfer efficiency in 293 cells plated in a 24-well dish. At 24 h after treatment, cells were lifted by trypsinization, washed, and counted by flow cytometry, measuring green fluorescence (GFP+: shaded; untreated cells: unshaded). In (A) and (B) 3PO was assembled and delivered to sub-confluent cells, as described [58]. (C) Capsid protein complexes are nontoxic relative to Ad5 on HeLa cells in culture. 1: untreated cells; 2: Ad5-GFP; 3: Protamine + DNA; 4: 3PO; 5: 3POF and 6: Fiber alone.

Figure 4. Protamine-mediated precipitation of 3PO

(A) 3PO was prepared by mixing 10 μg of DNA, 1 μg PBK10, and 10 μg protamine in a final volume of either 200, 100 or 50 μl. Mixtures were incubated at room temperature for 15 min, then spun in a microcentrifuge (15K RPM), while control samples were not. Supernatant absorbances were then measured at OD 260 to detect soluble DNA. (B) Increasing protamine excludes EtBr and forms precipitates. Plasmid DNA (0.35 μg, pEGFP) was incubated with indicated reagents for 15 min at room temperature, then electrophoresed on 0.8% agarose gel that was subsequently stained with EtBr to identify the DNA. (C) 3PO was prepared by mixing 0.35 μg PBK10, 3.5 μg DNA + increasing amounts of protamine in 100 μl final volume. After 15 min room temperature incubation, samples were spun in a micro-centrifuge (15K RPM), while control samples were not. Supernatant absorbances were then measured at 260 nm to detect soluble DNA. EtBr: Ethidium bromide.