Adenovirus serotype 5 (Ad5)-based vectors can bind at least three separate cell surface receptors for efficient cell entry: the coxsackie-adenovirus receptor (CAR), alpha nu integrins, and heparan sulfate glycosaminoglycans (HSG). To address the role of each receptor involved in adenoviral cell entry, we mutated critical amino acids in fiber or penton to inhibit receptor interaction. A series of five adenoviral vectors was prepared and the biodistribution of each was previously characterized in mice. To evaluate possible species differences in Ad vector tropism, we characterized the effects of each detargeting mutation in non-human primates after systemic delivery to confirm our conclusions made in mice. In non-human primates, CAR was found to have minimal effects on vector delivery to all organs examined including liver and spleen. Cell-surface alpha nu integrins played a significant role in delivery of vector to the spleen, lung and kidney. The fiber shaft mutation S*, which presumably inhibits HSG binding, was found to significantly decrease delivery to all organs examined. The ability to detarget the liver corresponded with decreased elevations in liver serum enzymes (aspartate transferase [AST] and alanine transferase [ALT]) 24 hr after vector administration and also in serum interleukin (IL)-6 levels 6 hr after vector administration. The biodistribution data generated in cynomolgus monkeys correspond with those data derived from mice, demonstrating that CAR binding is not the major determinant of viral tropism in vivo. Vectors containing the fiber shaft modification may provide for a detargeted adenoviral vector on which to introduce new tropisms for the development of targeted, systemically deliverable adenoviral vectors for human clinical application.

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During a toxicology study in cynomolgus (long-tailed or crab-eating) monkeys (Macaca fascicularis), a randomly distributed incidence of significantly increased hepatic enzyme activity was observed. Premedication hepatic enzyme activity in all monkeys of this study was normal, but increased alanine aminotransferase (ALT) activity was found in 4 of the 24 animals 2 weeks after initiation of the study and in 10 of 24 at 4 weeks. A drug-related effect was considered unlikely initially because the increases were not doserelated, and a 3-year review of 655 cynomolgus monkeys revealed a 15-20% incidence of increased hepatic enzyme activity. Good correlation was subsequently established between increased hepatic enzyme activity, active hepatitis A virus 0 infection, and histomorphologic confirmation of hepatitis (chronic periportal inflammation). Follow-up viral serodiagnostic screening of resident macaques revealed an overall incidence of anti-HAV IgG in 80% (155/193) of cynomolgus and in 70% (14/20) of rhesus monkeys. Serial screening demonstrated that several initially negative monkeys became seropositive for anti-HAV IgG, and a few acquired active infection (anti-HAV IgM). Among newly acquired cynomolgus monkeys, 2.5% (2/80) had an acute HAV infection, and 35% (28/80) eventually tested positive for anti-HAV IgG while quarantined in the primate facility. The characterization of an enzootic HAV infection in incoming monkeys posed a significant risk for the primate colony and handlers. Rigorous sanitation, isolation, and quarantine procedures, including personnel training and additional protective clothing for personnel working in the primate colony, reduced the potential for transmission and arrested the outbreak. Experimenters should be cautious in ascribing toxicity to a test article based solely on increased hepatic enzyme activity associated with chronic periportal inflammation.

An E1/E2a/E3-deficient adenoviral vector encoding an epitope-tagged (flagged) human factor VIII (FVIII) cDNA was delivered systemically to four cynomolgus monkeys. Analysis of liver biopsy samples revealed the presence of vector DNA at all points in the study (day 7, 28, and 56), with vector copy number declining approximately 10-fold between day 7 and day 56. Immunoprecipitation/Western analyses detected human flagged FVIII in the plasma of all monkeys and expression persisted for 14-28 days. Peak plasma FVIII levels ranged from 50 to 100 ng/ml. Bethesda assays revealed no inhibitor in two animals, the development of a low-level transient inhibitor in one animal, and an inhibitor titer that continued to increase for the duration of the study in one animal. Other treatment-related changes included modest increases in liver enzymes, an increase in interleukin-6 (IL-6) levels, and a transient decrease in platelets in all four animals. These data indicate that early generation adenoviral vectors do not support the long-term expression of FVIII in nonhuman primates.