Persistent hepatic expression of human apo A-I after transfer with a helper-virus independent adenoviral vector

Gene transfer with 'gutted' vectors is associated with persistent transgene expression and absence of hepatotoxicity, but the requirement of helper viruses hampers efficient production and leads to contamination of viral batches with these helper-viruses. In the present study, gene transfer with a helper-virus independent E-1/E-3/E-4-deleted adenoviral vector induced persistent expression of human apo A-I (200 16 mg/dl at day 35, 190 +/- 15 mg/dl at 4 months, 170 +/- 16 mgldl at 6 months) and stable transgene DNA levels (3.5 +/- 0.60 at day 35, 3.3 +/- 0.39 at 4 months, 3.1 +/- 0.47 mg/dl at 6 months) in C57BL16 mice in the absence of significant toxicity. The vector contained the 1.5 kb human alpha(1)-antitrypsin promoter in front of the genomic human apo A-I sequence and four copies of the human apo E enhancer (hAAT.gA-1.4xapoE) and was deleted in E-1, E-3 and E-4, Reintroduction of E-4 ORF 3 and E-4 ORF 4 in the viral backbone caused a more than four-fold decline of transgene DNA between day 35 and 4 months after transfer both in wild-type and in C57BL16 SCID and C57BL16 Rag-1(-/-) mice, indicating that the effect of E4 ORF 3 and E4 ORF 4 is independent of a cellular immune response against viral epitopes. Co-injection of an El-deleted vector containing no expression cassette and the E-1/E-3/E-4-deleted vector containing the hAAT.gA-1.4xapoE expression cassette indicated that E4 gene products destabilize transgene DNA in trans. Gene transfer with an E-1/E-3/E-4-deleted vector containing only E4 ORF 3 and the hAAT.gA-1.4xapoE expression cassette was associated with transgene DNA decline, but not with hepatotoxicity, indicating that transgene DNA persistence and hepatotoxicity are dissociated processes. After transfer with E-1/E-3/E-4-deleted vectors containing expression cassettes with a different promoter or a different position of the apo E enhancers, transgene DNA levels were less stable than after transfer with the vector containing hAAT.gA-1.4xapoE, indicating that the expression cassette is an important determinant of episomal stability. In conclusion, gene transfer with an E11EIE4-deleted vector containing the hAAT.gA-1.4xapoE expression cassette induces persistent expression of human apo A-I in the absence of hepatotoxicity. Transgene DNA turnover is independent of an adaptive cellular immune response against viral epitopes and of hepatotoxicity. E-1/E-3/E-4-deleted vectors containing transgenes under control of the hAAT promoter in combination with four copies of the human apo E enhancer may be suitable for hepatocytespecific overexpression of transgenes after gene transfer.