Genetic heterogeneity in response to adenovirus gene therapy

Abstract

Background: After intravenous delivery of the adenoviral vector into rats or mice, 95-99% of the encoded protein is produced in the hepatocytes. We observed, as have others, that the early expression levels of the vector encoded protein vary, greatly, within a species, from one animal strain to another. This study was initiated to determine the molecular mechanism causing the difference: hepatic transfection, transcription or translation. For this purpose different doses of Ad5 luciferase and Ad5 LacZ were intravenously injected into Brown Norway rats and Wag/Rij rats, two strains that differ by a factor of 10 in encoded protein levels. The proportion of LacZ positive hepatocytes, the adenoviral DNA, specific transgenic RNA and luciferase protein were compared in the two strains.

Results: The number of transduced hepatocytes and the amounts of Ad5 DNA in the livers was similar in both strains, whereas the Brown Norway rats produced 8 to 10 times more of both vector encoded proteins and of transgene mRNA than the Wag/Rij rats.

Conclusions: It is concluded that the difference between strains in vector encoded protein expression is due to different transcriptional events. No evidence was obtained to suggest that the differences are related to liver damage influenced by vector toxicity or immune reactions.

Figure 1

Plasma levels of ATF-BPTI in Brown Norway (black squares) and in Wag/Rij (open circle) after iv injection of 10¹⁰ iu Ad5 Adapt mhATF-BPTI. Ten animals per group, data are expressed as mean ± SD.

Figure 2

Plasma levels of endostatin in Brown Norway (black squares) and in Wag/Rij (open circle), after iv injection of 10¹⁰ iu Ad5 Adapt hEndostatin. Ten animals per group, data are expressed as mean ± SD.

Figure 3

Luciferase expression in the liver of the Wag/Rij and Brown Norway rats. A: luciferase activity determined 48 hours after iv injection of 10⁹ iu (gray bars, n = 6), 3 10⁹ iu (striped bars, n = 6), and 10¹⁰ iu (black bars, n = 12) Ad5 Adapt Luc. Data are expressed as mean ± SD.

Figure 4

Luciferase expression in the liver of different rat strains after iv injection of 10¹⁰ iu Ad5 Adapt Luc. The luciferase expression was determined two days (gray bars) and seven days (black bars) after virus injection. Determination after 2 or 7 days reveals a 10-fold difference (p = 0.0001, Scheffé post-hoc test.) in luciferase activity between the Wag/Rij and the Brown Norway. Six animals per group, data are expressed as mean ± SD.

Figure 5

The "porto-cava" gradient of the transgene expression viewed on a 50 × magnification field of liver section stained for X-Gal and counterstained with red. The livers were collected 2 days after iv injection of 10¹⁰ iu Ad5 Adapt LacZ A: Brown Norway rat. B: Wag/Rij rat. The white arrow points to a interlobular (portal) vein. The black arrow points to a central vein in the centro-lobular area.

Figure 6

Comparison of histological sections of the liver of Brown Norway and Wag/Rij rats 2 days after iv injection of 10¹⁰ iu Ad5 Adapt LacZ. The staining was carried out on 10-μm thick frozen sections. Four sections per slide, each slides represent one rat.

Figure 7

Northern Blots. Northern blot of the total RNA extracted from the liver of both rat strains. The hybridisation was done with a radioactive probe specific for the luciferase mRNA and then with a probe specific for the β-Actine mRNA. Lane 1–8: Wag/Rij rats, lane 1–2 animals injected with Ad5 Adapt Empty, lane 3–8: animals injected with Ad5 Adapt Luc. Lane 9–16: Brown Norway rats, lane 9–10 animals injected with Ad5 Adapt Empty, lane 11–16: animals injected with Ad5 Adapt Luc. The quantification of the signals with the phosphorimager shows that there is no difference in the quantity of β-actine transcript whereas there is about 10 times more luciferase transcripts in the liver of the Brown Norway rats than in the Wag/Rij rats. Means values +/- sd expressed in arbitrary units are depicted on the gel.