Pharmacokinetics of Adenoviral Vector-Mediated Gene Delivery to Vascular Smooth Muscle Cells: Modulation by Poloxamer 407 and Implications for Cardiovascular Gene Therapy

摘要

ABSTRACTRegionalin vivodelivery of therapeutic genes to the cardiovascular system at sites of localized vascular disease is feasible by catheter-mediated delivery of recombinant adenoviral vectors. Vascular smooth muscle cell (SMC) proliferation, which follows angioplasty and contributes to restenosis, is one process that may be amenable to such a gene therapy strategy. The clinical utility of localized delivery strategies such as this critically depends upon successful gene transfer to sufficient numbers of vascular cells, locally, within a clinically acceptable time period. Relatively limited information is available concerning the kinetics of gene transfer by first-generation, replication-deficient, recombinant adenovirus (Av1) vectors. In this context, we evaluated the pharmacokinetics of adenoviral vector-mediated gene delivery to vascular SMC using an Av1 reporter vector (Av1LacZ4) expressing a nuclear-targeted-galactosidase (-Gal) reporter. Bovine aortic SMC were exposed to Av1LacZ4 for various times at a range of concentrations and multiplicities of infection (MOI). After exposure, cells were washed and evaluated for transduction at 48 hr by X-Gal staining. Transduction occurred with a rate constant typically determined in the range of 1010to 1011eventsml/cellvirionmin. The rate of transduction was directly dependent on virion concentration, but not substantially on the virion-to-cell ratio. Relatively low fractions of the total input vector were found to be consumed, even after prolonged adsorption times. We hypothesized that the cellular transduction rate (and thus overall efficiency) would be improved by agents that could maintain a prolonged, high pericellular vector concentration. To evaluate this, cells were exposed to the vector in the presence of 15 grams/dl poloxamer 407, a viscous biocompatible polyol, for various times followed by washout and evaluation as described above. Both cells and vector remained viable under these conditions, and poloxamer was found to increase the apparent transduction rate 10-fold or more (15109transduction eventsml/cellvirionmin), with remarkable increases in numbers of cells transduced even after brief exposure periods. These observations demonstrate that the pharmacokinetics of adenoviral-mediated gene delivery to vascular SMC can be modulated by agents such as poloxamer 407, which may improve gene delivery by maintaining high pericellular concentrations of vector. Such modulation may permit achievement of desired levels of gene transfer while requiring lower total viral dosage and exposure time, and in turn may have important implications forin vivogene delivery to vascular tissues.