Gene transfer using liposome-complexed adenovirus seems to overcome limitations due to coxsackievirus and adenovirus receptor-deficiency of cancer cells, both in vitro and in vivo

Abstract

Use of adenoviruses as vehicle for gene therapy requires that target cells express appropriate receptors such as coxsakievirus and adenovirus receptor (CAR). We show here that CAR-deficiency in cancer cells, that limits adenoviral gene delivery, can be overcome by using adenovirus complexed with the liposome, Ad-PEGPE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]. We first confirmed that CT-26 mouse colon cancer cells are deficient in CAR by RT-PCR, and then showed that CT-26 cells infected with Ad-GFP/PEGPE exhibited highly enhanced expression of green fluorescent protein (GFP), compared with those infected with Ad-GFP. GFP expression depends on the dose of liposome and adenovirus. Luciferase expression in livers treated with Ad-luc/PEGPE was about 1,000-fold less than those infected with Ad-luc. In a liver metastasis mouse tumor model developed by intrasplenic injection of CT-26 cells, luciferase expression following i.v. injection of Ad-luc/PEGPE was significantly higher in tumors than in adjacent non-neoplastic liver. Following systemic administration of Ad-GFP/PEGPE, GFP expression increased in tumors more than in adjacent liver while the reverse was true following administration of Ad-GFP. In the latter case, GFP expression was higher in liver than in tumors. This study demonstrates that systemic delivery of PEGPE-adenovirus complex is an effective tool of adenoviral delivery as it overcomes limitation due to CAR deficiency of target cells while reducing hepatic uptake and enhancing adenoviral gene expression in tumors.

Figure 1

Expression of CAR and αν integrin in cancer cell lines after Ad-GFP treatment. (A) Expression levels of CAR and αν integrin mRNA transcripts in mouse cancer cell lines. Total RNAs were isolated from each cell lines and RT-PCR was performed with mouse GAPDH as a control. PCR products were separated on 1.2% agarose gels and stained with ethidium bromide. (B) Adenoviral gene expression in CAR-positive and -negative cell lines. Mouse cancer cells were seeded in six-well plates and infected with 30 MOI of Ad-GFP to Hepa1-6 cells or 200 MOI of Ad-GFP to CT-26, NIH3T3, and B16F10 cells. After 1 day, GFP expression in each cells was determined at 40× magnification taken using fluorescent microscope.

Figure 2

Effect of liposome dose and adenovirus concentration on efficacy of liposome-complexed adenoviral gene expression. (A) Effect of liposome dose on adenoviral gene expression in CT-26 cells. Varying amounts of PEGPE liposome were incubated with concentration of Ad-GFP (1 × 10² pfu/µl) for 30 min and then added to CT-26 cells. After 24 h, the percentage of GFP expressing cells was determined by FACS analysis. The data represents mean ± S.D. of triplicate experiments. (B) Effect of adenovirus concentration. Varying concentrations of Ad-GFP were incubated with PEGPE liposome at fixed liposome dose (2.5 µl). The next experimental procedures were the same as (A).

Figure 3

Tissue distribution of liposome-complexed adenovirus in normal mice after systemic administration. (A) Luciferase expression in liver following systemic administration of adenoviruses. 1 × 10¹¹ vp of Ad-luc or Ad-luc/PEGPE was injected intravenously and transgene expression was measured after 24 h. Liver was harvested, homogenized and then luciferase activity was measured using the kit according to the manufacturer's instructions. Each data represents the mean ± S.D. (n = 5). (B) Tissue distribution of luciferase expression following systemic administration of adenoviruses. 1 × 10¹¹ vp of Ad-luc or Ad-luc/PEGPE was intravenously injected into the Balb/c mice and luciferase expression was measured in each tissue. Luciferase activity in each tissue was calculated relative that in the liver. The data represent the mean ± S.D.

Figure 4

Expression of liposome-complexed adenovirus in liver metastasis tumor model. (A) GFP expression in intrahepatic tumors (T) and adjacent liver following systemic administration of adenoviruses. 1 × 10¹¹ vp of Ad-GFP or Ad-GFP/PEGPE was injected intravenously and livers, including tumors were removed after 24 h. Eight-µm frozen sections were prepared and mounted with cover glass. Fluorescence was observed by Olympus fluorescent microscope (×40). After then, the slides were stained with hematoxylin and eosin, and observed under the light microscope (×40). (B) Relative ratio of luciferase expression of intrahepatic tumors to adjacent non-neoplastic liver following systemic administration of adenoviruses. 1 × 10¹¹ vp of Ad-luc or Ad-luc/PEGPE was intravenously injected and luciferase expression was measured in tumors and adjacent normal livers. Luciferase activity in tumors was calculated by relative to that in adjacent non-neoplastic liver. Data represents the mean ± S.D.