Adenovirus-mediated regulable target gene expression in vivo

Abstract

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.

Figure 1

(A) Regulatory system. The regulator GLVPc′ consists of a mutated human progesterone receptor–LBDΔ, a DNA binding domain of yeast GAL4 (GAL4–DNA binding domain), and an activation domain of herpes simplex virus (VP-16). Regulator GLp65 contains the activation domain of p65. The target consists of four GAL4 binding sites and a TATA-box linked to the luciferase reporter gene. Luc, luciferase; PR, progesterone receptor. (B) Mifepristone-dependent target gene induction by GLVP compared with GLp65. GLVPc′ or GLp65 (0.3 μg per well on a 6-well plate) were transiently transfected in Hela cells with the 17 × 4-TATA-luciferase as a reporter (0.3 μg per well on a 6-well plate) (B) by using 17 × 4-tk-luciferase as a reporter (C). The luciferase activity is shown as relative luciferase units (RLU). Control, transfection of the reporter and expression vector backbone; +, presence of mifepristone (Ru 486) [10⁻⁸]; −, absence of mifepristone (Ru 486). Error bars show standard deviation.

Figure 2

Structure of hGH-GLp65 and hGH-H-GLp65. The constructs contain the left terminus of adenovirus type 5 (nucleotides 1–440), a 16,054-bp fragment of the human hypoxanthine-guanine phophoribosyltransferase gene, a regulatory cassette containing the regulator GLp65. UAS-TATA-GH, hGH under upstream activation sequence–TATA control; 2 × HS4, insulator, a 5′element of the chicken β-globin domain (from G. Felsenfeld); TTRB, liver-specific promoter enhancer (from R. Costa); GLp65, inducible gene switch p65 activation domain; SV40, poly(A), the 6,545-bp fragment out of the C346 cosmid and the right terminus of adenovirus type 5 (nucleotides 35,818–35,935). HGH-H-GLp65 contains an insulator sequence; hGH-GLp65 does not.

Figure 3

Induction of hGH on adenoviral transduction. (A) C57BL/6 mice (8–10 weeks old) were infected in the tail vein at day 0 with 1 × 10⁹ infectious particle units of hGH-GLp65. Mifepristone (Ru 486) (250 μg/kg) was administered every second day after infection for a period of 2 weeks as indicated by arrows. Mice were bled at different time points, and serum hGH was analyzed by radioimmunoassay. Mice 1 and 2 received i.p. injections of mifepristone; mouse 3 (minus mifepristone) received sesame oil. hGH serum levels are shown micrograms per milliliter. (B) Kinetics of inducing hGH in mice 2 weeks after adenoviral infection. Mice infected for 2 weeks with the regulable adenoviral construct hGH-GLp65 were induced with 500 μg/kg mifepristone as indicated by an arrow. Blood was drawn from the mice 3, 6, 12, 24, 48, 72, 120, and 192 h after mifepristone (Ru 486) administration, and hGH was measured in the serum by a radioimmunoassay. hGH serum levels of individual mice are shown in micrograms per milliliter.

Figure 4

Repetitive induction of hGH in transduced mice. Mice infected with hGH-GLp65 or hGH-H-GLp65 adenoviral vectors were induced three times with 250 μg/kg mifepristone over a time period of 50 days. hGH was measured before, 12 h after, and 7 days after mifepristone administration. Graph shows independent mice that received mifepristone (+) or just carrier as a control (−). Serum levels of hGH are shown in micrograms per milliliter.

Figure 5

Long-term expression of hGH in transduced mice. Mice infected with hGH-GLp65 or hGH-H-GLp65 received, 4 weeks after infection, biodegradable pellets (360 μg/pellet, released in 60 days) by transplantation containing mifepristone (+) or carrier (−) only. Mice were weighed, and blood was drawn 3, 13, 20, and 27 days after drug administration. (A) hGH levels (micrograms per milliliter). The numbers of mice for each construct is three; bars show the standard error. (B) Weight of the mice (grams).