138. Blood Factors Affect Adenovirus Infectivity and Bio-Distribution In Vivo
Authors: Dmitry M. Shayakhmetov
Journal: Molecular Therapy (2005)

An incomplete understanding of adenovirus (Ad) interactions with host factors has significantly hampered application of Ad-based vectors for human gene therapy. This is particularly true following intravascular delivery, when the bulk of the vector dose is rapidly cleared from the blood and transduction of liver tissue and innate immune responses contribute to the overall poor performance of Ad as a therapeutic vector. Accumulating data suggest that liver-mediated Ad clearance from the blood and Ad infection of liver cells do not occur through the classical model of Ad infection, which relies on the presence of the coxsakie- and adenovirus receptor, CAR, and alpha-v integrins. Both, CAR and/or integrin binding-ablated vectors exert dramatic innate immune responses and are rapidly cleared by the liver upon systemic application. Recently, we discovered that blood coagulation factor IX directly binds to the Ad fiber knob domain and mediates hepatocyte and Kupffer cell infection in vivo through interaction with novel hepatocellular receptors. Our data, however, indicate that in addition to FIX, other blood factors are likely to contribute to Ad liver cell infection. To study in a systematic way host factors interacting with Ad upon intravascular application, we incubated freshly purified mouse plasma with recombinant Ad5 or Ad35 fiber knob domains immobilized on agarose beads, and Ad fiber knob-bound proteins were recovered and analyzed by tandem mass spectrometry. These analyses revealed that multiple plasma proteins efficiently bind to the Ad fiber knob domain. We confirmed that most of them (including alpha-2 macroglobulin, fibrinogen, complement C3, and complement C4-binding protein, C4BP) are capable of direct binding to Ad fibers in a slot-blot assay. To investigate whether any of the identified Ad-binding plasma proteins can mediate liver cell infection, we infected human hepatocarcinoma cells with an Ad5F|[ast]| vector, possessing a single point mutation abrogating its binding to CAR. While supplementation of Ad5F|[ast]| virus containing media with alpha-2 macroglobulin, fibrinogen or complement C3 resulted in very low background levels of infection, supplementation of media with C4BP completely restored Ad5F|[ast]| infectivity toward these cells. Additionally, we confirmed that C4BP-mediated infection was dose dependent, mediated by the fiber knob domain, and could be efficiently competed with ligands that bind heparan sulfate proteoglycans and LDL-related receptor protein, LRP. C4BP is one of the most abundant plasma proteins, present at an average concentration of 250 |[mu]|g/ml. Clearly, upon intravascular administration, Ads will be exposed to large amounts of C4BP capable of targeting them to liver cells. While further studies should show whether Ad binding to other blood factors is of any physiological significance, our data indicate that there is a complex network of interactions between Ad and plasma proteins, that contribute to the observed pharmacokinetics, bio-distribution and toxicity after intravascular application. Our study helps better understand Ad-host interactions in vivo, when vector applied systemically. It also provides the basis for developing rational approaches toward construction of safe and efficient Ad vectors for human gene therapy applications.