889. Lanthanum Enhances Adenovirus-Mediated Gene Transfer

Abstract

Top of pageAbstract Introduction: In this study we investigated the use of lanthanum for improved efficiency of adenoviral gene transfer. Lanthanum ions, La 3+, are stable inorganic ions with a high charge to volume ratio enabling them to bind to negatively charged proteins with high efficiency. Methods: First generation adenoviruses encoding human Bone Morphogenetic Protein-2 (Ad-BMP-2) or Green Fluorescence Protein (Ad-GFP) cDNA were used. Osteoprogenitor cells were obtained from Osteogenesis Imperfecta Mice (OIM). La3+/virus complexes were prepared by adding the vectors to serum-free DMEM. LaCl3 was then added (200 |[mu]|M). Viral doses ( 10-300 MOI) were used for infection in 24-well plates. Non-lanthanum groups contained adenoviral vector and serum-free DMEM only. The viral mixtures were added to OIM cells and incubated. OIM cells in 24 well cell culture plates were infected with Ad-GFP with and without Lanthanum. (MOI = 300, 100, 30, 10). After 48 h, the samples were analyzed on FACScan. The induction of Alkaline Phosphatase Activity was assessed at 5 days. Results: Flow Cytometry showed, that Lanthanum enhanced transduction efficiency for all viral doses. The effect was highest at 10 MOI (12-fold increase) and diminished slightly with increasing viral dose (Fig. 1). The mean fluorescence intensity within transduced cells also increased in all lanthanum groups (Fig.2). However this effect decreased dramatically with viral dose, ranging from 18-fold at 300 MOI to only 3-fold at 10 MOI. To determine if La-Ad complexes could enhance a biological response, BMP-2 induction of Alkaline Phosphatase Activity was measured in OIM cells in the presence and absence of La3+. Alkaline phosphatase activity increased dose-dependently with Ad.BMP-2 infection, with a peak activity of 310 U/L at 100 MOI. Addition of La3+, increased alkaline phosphatase activity at 10 MOI and 30 MOI compared to virus only, suggesting enhanced adenoviral-mediated expression of BMP-2 at these doses. Moreover, equivalent levels of alkaline phosphatase activity, |[sim]|300 U/L, could be generated with 3-fold less viral vector in the presence of La3+. Discussion: Our findings indicate that the addition of lanthanum to adenoviral vectors can increase their transduction efficiency. This may allow for a reduction in the number of adenoviral particles, reducing viral production costs and immunological burden of virus.