Adenoviral vectors Research Papers - Academia.edu (original) (raw)

Adenoviral vectors are among the most commonly used vectors in gene therapy. Unfortunately, several adverse side effects complicate treatment with these vectors, including thrombocytopenia. Recent advances in vector design have led to the development of vectors that lack many nonessential adenovirus genes (gutless vectors), resulting in considerable reduction in side effects such as acute hepatitis. In spite of these advances, thrombocytopenia remains an important complication of adenoviral gene therapy, suggesting that its mechanism may not require the expression of virus-specific genes. To gain a better understanding of the mechanisms involved in adenovirus-induced thrombocytopenia, we investigated the possibility of a direct interaction between the adenoviral vectors and platelets. Flow cytometry experiments using a fluorescein isothiocyanate (FITC)-labeled anti-adenovirus monoclonal antibody revealed binding of the adenovirus to human platelets in a dose-dependent manner. Bound virus was detectable at a 1,000:1 ratio of viral particles to platelets with a substantial increase in binding observed when the ratio was increased to 2,000:1. When added to human platelet-rich plasma, adenoviruses activated
the platelets, as evidenced by an increase in the expression of both P-selectin and active alphaIIbbeta3 receptors, detected with the monoclonal antibodies CLBThromb/6 and PAC-1, respectively. Adenovirus-activated platelets also bound FITC-fibrinogen. We also examined the effect of adenovirus on protein tyrosine phosphorylation, both ex vivo in human platelets, and in vivo in mouse platelets. In both instances, adenovirus vectors (both first generation and gutless) induced dose- and time-dependent tyrosine phosphorylation of a number of platelet proteins.
Among these, we have tentatively identified PI3-kinase, rac, rho, FAK and MAP kinase. In studies of the time-course of thrombocytopenia in mice, we observed the platelet count to drop to approximately 20% of pretreatment values at 1hr. We then studied whether alphaIIbbeta3 blockade might ameliorate the thrombocytopenia. When the small molecule alphaIIbbeta3 inhibitor, tirofiban, was administered simultaneously with the adenoviral vector, the decrease in platelet count was markedly blunted, being reduced only to 80% of pretreatment values. In summary, we have shown that adenovirus vectors used in gene therapy bind to and activate platelets, inducing tyrosine phosphorylation of a large number of platelet signaling proteins. The thrombocytopenia induced by these vectors was markedly attenuated in mice treated with the alphaIIbbeta3 inhibitor tirofiban, suggesting a therapeutic strategy for preventing thrombocytopenia in patients treated with these vectors.