Construction and Radiolabeling of Adenovirus Variants that Incorporate Human Metallothionein into Protein IX for Analysis of Biodistribution

Abstract

Using adenovirus (Ad)-based vectors is a promising strategy for novel cancer treatments; however, current tracking approaches in vivo are limited. The C-terminus of the Ad minor capsid protein IX (pIX) can incorporate heterologous reporters to monitor biodistribution. We incorporated metallothionein (MT), a low-molecular-weight metal-binding protein, as a fusion to pIX. We previously demonstrated 99mTc binding in vitro to a pIX-MT fusion on the Ad capsid. We investigated different fusions of MT within pIX to optimize functional display. We identified a dimeric MT construct fused to pIX that showed significantly increased radiolabeling capacity. After Ad radiolabeling, we characterized metal binding in vitro. We explored biodistribution in vivo in control mice, mice pretreated with warfarin, mice preimmunized with wild-type Ad, and mice that received both warfarin pretreatment and Ad preimmunization. Localization of activity to liver and bladder was seen, with activity detected in spleen, intestine, and kidneys. Afterwards, the mice were euthanized and selected organs were dissected for further analysis. Similar to the imaging results, most of the radioactivity was found in the liver, spleen, kidneys, and bladder, with significant differences between the groups observed in the liver. These results demonstrate this platform application for following Ad dissemination in vivo.

Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusion

“…It is also clear that this is much less of a problem during Zn(II) metalation because of the decreased pH sensitivity at physiological pH. MT also binds a number of exogenous heavy metals involved in chemotherapeutics [81][82][83] and water contamination 84 and the pH considerations raised in this paper likely apply to other MT-metal binding systems. Similar metalation experiments with human MT2a show a mixed mechanism at pH 7.4 30 and rabbit liver MT2a has been shown by NMR to cluster at pH 7.2, with the signal disappearing at higher pH.…”

Targeted Adenoviral Vectors I

“…145 Using this strategy, Ad retargeting was achieved by incorporating large targeting moieties, including eGFP, 146 HSV1-tk, 147 and metallothionein. 148,149 The use of pIX protein as a platform for presenting scFv or sdAb molecules for Ad retargeting was evaluated. The 13R4 scFv directed against β-galactosidase, which was selected for its capacity to fold correctly in a reducing environment such as the cytoplasm, was fused with pIX using a 75-Å-spacer sequence.…”

Fusion of Large Polypeptides to Human Adenovirus Type 5 Capsid Protein IX Can Compromise Virion Stability and DNA Packaging Capacity

“…The human, canine, and bovine pIX were shown to tolerate the addition of large polypeptides, including red, yellow, or green fluorescent proteins (18)(19)(20)(21)(22)(23). pIX can also accommodate large polypeptides that can aid in determining virus biodistribution, such as human metallothionein, herpes simplex virus thymidine kinase, or firefly luciferase protein (24)(25)(26). pIX has served as a site for addition of a biotin acceptor peptide for metabolic biotinylation of the HAdV capsid, allowing for the subsequent avidin-mediated purification of the virus (27), or addition of avidin-linked targeting ligands (28).…”