Characterization of hemodynamic events following intravascular infusion of recombinant adenovirus reveals possible solutions for mitigating cardiovascular responses.

Intravascular administration of recombinant adenovirus (rAd) in cancer patients has been well tolerated. However, dose-limiting hemodynamic responses associated with suppression of cardiac output have been observed at doses of 7.5 x 10(13) particles. While analysis of hemodynamic responses induced by small-molecule pharmaceuticals is well established, little is known about the cardiovascular effects of rAd. Telemetric cardiovascular (CV) monitoring in mice was utilized to measure hemodynamic events following intravascular rAd administration. Electrocardiogram analysis revealed a block in the SA node 3-4 min postinfusion, resulting in secondary pacemaking initiated at the AV node. This was associated with acute bradycardia, reduced blood pressure, and hypothermia followed by gradual recovery. Adenovirus-primed murine sera with high neutralizing antibody (nAb) titers could inhibit CV responses, whereas human sera with equivalent nAb titers induced by natural infection were, surprisingly, not inhibitory. Interestingly, repeat dosing within 2-4 h of the primary injection resulted in desensitization, resembling tachyphylaxis, for subsequent CV responses. Last, depletion of Kupffer cells prior to rAd infusion precluded induction of CV responses. These inhibitory effects suggest that rAd interactions with certain cells of the reticular endothelial system are associated with induction of CV responses. Significantly, these studies may provide insight into management of acute adverse effects following rAd systemic delivery, enabling a broadening of therapeutic index.

Characterization of empty capsids from a conditionally replicating adenovirus for gene therapy.

As virus vectors for gene therapy approach the goal of successful clinical treatment, it is increasingly necessary for the product to be fully characterized. Empty capsids are perhaps the main extraneous component of recombinant adenovirus (rAd) products that are purified by column chromatography. Two diverse rAd products, one a replication-defective rAd and the other a conditionally replicating rAd, show different protein compositions of their empty capsids. The empty capsid type from the replication-defective rAd carrying the gene for p53 was previously determined to have approximately 1400 copies per particle of pVIII, the precursor to the hexon-associated protein VIII (Vellekamp et al., Hum. Gene Ther. 2001;12:1923-1936). Quantification of this protein is a useful measure of the amount of empty capsids in preparations of this vector. Here we purify and characterize empty capsids from the conditionally replicating rAd. This empty capsid type lacks any appreciable amount of pVIII but contains pVI and multiple forms of the L1 52/55K protein, mostly as disulfidelinked oligomers. Empty capsid from conditionally replicating rAd present new challenges in terms of its quantification, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis densitometry analysis suggests that the amount of this empty capsid in a preparation, like that of rAd p53 empty capsid, declines with increased time of infection. This empty capsid demonstrates heterogeneity by anion-exchange chromatography, electron microscopy, and CsCl density gradient centrifugation.