Gene Therapy advance online publication 15January2015; doi: 10.1038/gt.2014.124

First-generation (E1 deleted) adenovirus vectors (FG AdVs), which lack the E1 and E3 regions, are popularly used in basic studies to elucidate gene functions, and have been employed for gene therapy.1, 2, 3, 4 Because the DNA fragments of up to about 7 kilobases (kb) in total can be inserted into the AdV genome, the AdVs are frequently used to produce two proteins simultaneously from two independent transgenes expressing both the target gene and the reporter gene, for example. In the studies using the cultured cells and in the animal experiments, the GFP and luciferase are used as the reporters. Recently, positron emission tomography has clinically been used in patients for diagnoses and in experimental animal models. Therefore, the AdVs containing both the therapeutic gene and the positron emission tomography reporter gene would be valuable in the gene therapy fields, because the therapeutic effects, the vector duration and distribution can simultaneously be monitored.5, 6, 7, 8 Probably one would wish for high-titer AdVs with the highest expression for the therapeutic gene and with the second highest for the reporter gene not causing any trouble, if the insertion sites and orientations in the AdV genome can be chosen. However, the titers and the expression levels of the AdVs may considerably be influenced by the sites and orientations of the transgenes. Such information may be very valuable for construction of the best vector, especially in the vector containing both the therapeutic gene and the reporter gene.

The simultaneous expression of two genes could be achieved by inserting the two genes into the E1 site under the control of a single prompter using the internal ribosomal entry sites or using porcine teschovirus-1 2A.9, 10 In the former approach, the expression of the second gene might be influenced by the sequences between internal ribosomal entry sites and its initiation codon, and in the latter, the manipulation is necessary to remove the stop codon of the first gene and to adjust the frames of the two genes. When two genes driven by the independent promoters are inserted into the E1 site, they might interfere with each other. However, when two independent expression units are inserted in different sites in the AdV genome, no interference occurs. Moreover, the advantage of this approach is that the main target gene can easily be changed using the AdV cassette that already contains the reporter gene.

There are three insertion sites and two orientations: a transgene can be inserted into the AdV genome by substitution of the E1 or E3 gene and by simple insertion at a position upstream of the E4 gene. Therefore, there are six different possible sites/orientations for any given transgene. Moreover, not only the potent promoters such as EF1 but also tissue-specific promoters such as -fetoprotein (AFP) can also be employed. Although the studies examining which sites/orientations are superior to others are practically important, they have been very limited11, 12 and systematic analyses have not been reported so far.

As it is known that the expression level of a transgene varies considerably depending on the site in the cell chromosome of the human genome, the phenomenon is called the position effect.13, 14 Although CG-methylation in the cell chromosome is clearly one reason, it is not observed in the AdV genome. Therefore, it would be of interest to examine whether the position effect might also be observed similarly in the AdV genome for the potent promoter and for the tissue-specific promoter.

FG AdVs retain almost all viral genes. They are normally not expressed in the target cells, because E1A protein, the essential transactivator for expression of all other viral genes, is not present. However, there is one report of splicing of aberrant mRNAs from the inserted foreign genes to a viral gene.15 In this case, the aberrant mRNAs are transcribed by strong foreign promoters and produce transgene-viral gene fusion proteins, which elicit strong immune responses. However, it is not known whether the production of the aberrant gene product between the inserted transgene and viral gene is rare or not.

In this study, we examined the AdV titers and expression levels of an identical transgene inserted at the E1, E3 and E4 sites. We used three transgenes, namely, GFP, LacZ and Cre, and two promoters, namely, the potent EF1 promoter and the cancer-specific AFP promoter, and attempted to construct AdVs using all combinations, that is, 18 AdVs, and succeeded in constructing 17 of them. We found that insertion at the E1 and E4 sites yielded mostly high titers, whereas the one at the E3 yielded variable titers. Surprisingly, four aberrantly spliced mRNAs between the transgenes and viral genes were found in the vector obtained by insertion at the E3 site, which was probably the reason for the very low titers. As for the expression levels, clear differences were observed among the vectors obtained with insertion at the E1, E3 and E4 sites despite using the identical transgene, indicating that the position effect was certainly present for the AdV genome and that aberrant splicing may, at least in part, explain this effect. We also propose a strategy to avoid generation of the aberrantly spliced mRNAs.

We first examined whether the vector titers were influenced by the site/orientations of the transgenes containing a potent EF1 promoter. Towards this end, we attempted to construct six GFP-expressing (EF-GFP) and six LacZ-expressing (EF-LacZ) vectors in all possible combinations, that is, the E1, E3 and E4 insertion sites and the two orientations ( Figure 1), and measured the vector titers (Figure 2a) (hereinafter, the vectors will be designated as per the following; the vectors containing the GFP gene and LacZ gene at the E1 insertion site and in the left orientation shall be denoted as G-E1L and Z-E1L vectors, respectively). Among the GFP-expressing vectors, high titers were obtained for G-E1L, G-E3L, G-E4L and G-E4R vectors (Figure 2a, bars 1, 3, 5 and 6), while the titer for the G-E1R vector was lower (bar 2). Notably, the G-E3R vector, that is, vector with the GFP transgene inserted in the E3 site in the rightward orientation, could not be obtained despite three independent attempts (bar 4, denote ). Therefore, although exactly the same EF1-GFP expression unit was inserted in these vectors, the sites and orientations exerted considerable influence on the vector titers and even determined whether the vector was available or not. Similar results were obtained for vectors expressing LacZ: the titers of the Z-E1L, Z-E4L and Z-E4R vectors (bars 7, 11 and 12) were high, and that of the Z-E1R vector was also low (bar 8). However, the results of insertion at the E3 site differed for GFP and LacZ. The titer ratio of Z-E3L was significantly lower than that of G-E3L (compare bars 3 and 9, described later), and the Z-E3R vector was available, although its titer was extremely low (bar 10). Therefore, the GFP gene and LacZ gene themselves influenced the vector titers.

The FG AdV structures of six different site/orientations in all possible combinations. The box containing pro, gene and pA represents the expression unit and the arrows show the orientation of transcription. pro, EF1 and AFP promoter; gene, GFP, LacZ and Cre; pA, rabbit -globin polyadenylation signal. For example, the vector containing the transgene at the E1 insertion site and in the left orientation is denoted as E1L.

Titers of the virus vectors containing identical expression units. (a) Virus titers of the AdVs containing the EF1 promoter. The AdV genomes transduced into the HuH-7 cells were measured 3 days post infection. The virus titers were calculated relative to the copy numbers of the AdVs.16 The titer of the E1L vector was set as 1; G-E1L, 8.3 108 relative virus titer (rVT)/ml, L-E1L, 5.0 109 rVT/ml. indicates that G-E3R could not be obtained. (b) The titers of the virus vector containing Cre gene driven by the AFP promoter. E1L vector was used as the control. *P<0.05, **P<0.01.

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Gene Therapy - Preferable sites and orientations of ...