Variables affecting in vivo performance of high-capacity adenovirus vectors

Abstract

In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.

FIG. 1.

Schematic structure of HC-Ad vectors and Ad vectors lacking E1. The HC-Ad vectors have deletions of all viral genes and contain only the left and right termini of Ad5. In addition, AdSTK109 and AdSTK135 contain the genomic hAAT gene locus, including the liver- and macrophage-specific promoters. AdVB8.AAT contains the genomic hAAT promoter, the first hAAT exon and intron, the splice acceptor of the second intron, and the hAAT cDNA. AdGS47, AdGS61, AdGS67, and AdGS86 contain the murine pgk promoter and hAAT cDNA. In contrast to AdGS47, AdGS61, and AdGS86, which carry stuffer DNA derived from HPRT and C346, AdGS67 contains lambda DNA as the stuffer. AdGS85 contains the hCMV promoter and the hAAT cDNA plus HPRT and C346 stuffer. In addition, the HC-Ad vectors bear presumptive MARs located originally either in the HPRT stuffer or in the first intron of the hAAT gene locus. AdGS84 is an Ad vector lacking E1 which contains the murine pgk promoter and hAAT cDNA. The arrows indicated the transcriptional orientation of the hAAT expression cassette.

FIG. 2.

Effects of using the genomic locus or cDNA on the expression of hAAT. Groups of 6 C57BL/6J mice were injected with 10⁹ infectious units of either AdSTK109 #39, AdVB8.AAT, or AdGS47 in the tail vein. The amounts of hAAT in serum at the indicated time points were determined by ELISA. Error bars represent the standard deviations of each group.

FIG. 3.

Influence of stuffer DNA from different origins (human and bacteriophage lambda) on transgene expression. Groups of 6 C57BL/6J mice were injected with either 10⁹ infectious units of AdGS47 (HPRT, C346 stuffer) or 2 × 10⁹ infectious units of AdGS67 (lambda stuffer) in the tail vein. The amounts of hAAT in the sera of transduced mice were analyzed by ELISA. Error bars represent the standard deviations of each group. Please note that the data for AdGS47 are the same as shown in Fig. 2.

FIG. 4.

Influence of MARs on transgene expression. Groups of 6 C57BL/6J mice were injected with 10⁹ infectious units of AdSTK109 #39 (containing the HPRT MAR) or AdSTK135 (HPRT MAR replaced by lambda sequences) (A) or AdGS47 (containing the HPRT MAR) or AdGS61 (containing the HPRT and hAAT MAR) (B) in the tail vein. The amounts of hAAT in the sera of mice at the indicated time points were determined by ELISA. Error bars represent the standard deviations of each group. Please note that the data for AdSTK109 #39 and AdGS47 are the same as shown in Fig. 2.

FIG. 5.

Effects of location and orientation of the transgene cassette on hAAT expression. Groups of 6 mice were injected with 10⁹ infectious units of AdGS86 #9, AdGS86 #20 (expression cassette in different orientations located at the left terminus), or AdGS47 (expression cassette flanked by HPRT and C346 stuffer) (A) or AdSTK109 #35 or AdSTK109 #39 (expression cassette in different orientations) (B) in the tail vein. The amounts of secreted hAAT in the sera of transduced mice were determined by ELISA. Error bars represent the standard deviations of each group. Please note that the data for AdSTK109 #39 and AdGS47 are the same as shown in Fig. 2.

FIG. 6.

Expression of hAAT in mice transduced with Ad vectors lacking E1. Groups of 6 mice were injected with 10⁹ infectious units of either AdGS84 #15 or AdGS84 #17 (expression cassette in opposite orientation) in the tail vein, and the amounts of hAAT in the serum at different time points were determined by ELISA. Error bars represent the standard deviations of each group.

FIG. 7.

Influence of the promoter on antibody generation. Six C57BL/6J mice were injected with 10⁹ infectious units of AdGS85, which carries the hAAT cDNA expressed from the hCMV promoter. Mice were bled at the indicated time points, and levels of hAAT in the serum (A) or titers of antibodies to hAAT (B) were determined by ELISA.