Toxicological Comparison of E2a-Deleted and First-Generation Adenoviral Vectors Expressingα1-Antitrypsin after Systemic Delivery

Abstract

Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human α₁-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 × 10¹² particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity. To evaluate any potential safety advantage of E2a-deleted adenoviral vectors, we have compared in vivo first-generation (deleted in E1) and second-generation (deleted in E1 and E2a) vectors containing the human α₁-antitrypsin gene. Toxicity was evaluated for a period of 21 day after intravenous administration of increasing doses of each vector in C3H mice. The E2a-deleted vector was found to be slightly less toxic at an intermediate dose of 3 × 10¹² particles/kg. However, most parameters measured did not differ between the two vectors, and the slight decrease in toxicity with the E2a-deleted vector was offset by a decreased level of transgene expression per particle. We conclude that new vectors may need to be developed to improve further the therapeutic index.