Recombinant adeno‐associated virus‐mediated TRAIL gene therapy suppresses liver metastatic tumors

Abstract

To evaluate the tumoricidal activity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on disseminated liver metastatic tumors, we constructed a recombinant adenoassociated virus (rAAV) expressing the extracellular domain (95-281aa) of human TRAIL (TRAIL , and the recombinant virus was designated as rAAV-TRAIL) using the 3-plasmid, helper-virus-free, packaging system. Transduction of mouse lymphoma EL-4 cells and Jurkat T cells lead to the expression of TRAIL 95-281 protein in both virus-transduced cells and the culture media, along with apoptosis of these cells in vitro. The therapeutic potential of rAAV-TRAIL was then evaluated in an orthotopic transplanted mouse model mimicking liver cancer metastasis, which was established by injection of EL-4 cells into the liver of C57BL/6 mice via the hepatic portal veins. Subsequent intraportal vein injection of rAAV-TRAIL, not the control virus, into the liver of these mice resulted in significant suppression of tumor growth and prolonged survival, while normal hepatocyte toxicity is undetectable. Histological and biochemical analysis in tumor tissue and serum confirmed that TRAIL 95-281 was stably expressed in relatively high level in hepatocytes and was secreted into the serum in active trimeric form. Futhermore, the mechanism for rAAV-TRAIL to inhibit tumor growth was by inducing apoptosis of the tumor cells metastasizing to the livers. These results strongly suggest that the rAAV-TRAIL-mediated gene delivery could be a promising approach for the treatment of liver metastasis cancer. ' 2005 Wiley-Liss, Inc.Key words: adeno-associated virus vector; tumor necrosis factorrelated apoptosis inducing ligand; EL-4 cells; liver metastasis; gene therapy Metastasis is the major cause of cancer therapeutic failure and patient mortality. The most frequent site of blood-borne metastases is the liver, which is also involved in about one third of all cancers.1 The liver metastasis of lymphoma universally occurs in the clinical settings, resulting in poor prognosis in patients. Resection of liver metastasis constitutes the only curative treatment but is only feasible for about 10% of all patients with high recurrence rate. Chemotherapy and embolization are at best palliative but have no impact on survival or life-span. Therefore, a substitutional therapeutic strategy for the treatment of metastatic liver cancers is exigently sought.

Oral adeno-associated virus-sTRAIL gene therapy suppresses human hepatocellular carcinoma growth in mice

“…Infectious AAV particles were generated in human embryo kidney cells with packaging-helper plasmid pDG 12 and purified via heparin affinity chromatography as previously described. 9 The recombinant virus titers were determined by measuring the copy number of viral genomes in pooled, dialyzed gradient fractions using a real-time PCR (RT-PCR) procedure with primers of the WPRE enhancer region. The standard titer was determined via preexperiment of SMMC-7721 cells transduced with AAVenhanced green fluorescent protein (EGFP).…”

“…cDNA was prepared via reverse transcriptional PCR as previously described. 9 The relatively quantitative PCR reaction was set up in a total volume of 20 L containing 2 L of diluted cDNA, 10 L of 2ϫ SYBR Green PCR mix buffer (ABI, Foster City, CA), and 1 mol/L of each primer. RT-PCR was performed by using WPRE primers 5Ј-TGGCGTGGTGTGCACTGT-3Ј and 5 Ј-GTTCCGCCGTGGCAATAG-3Ј and ␤-actin primers 5Ј-ATGGTGGGAATGGGTCAGAAG-3Ј and 5Ј-CACGCAGCTCATTGTA GAAGG-3Ј on Applied Biosystems 7500 (ABI) at 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.…”

“…This result is consistent with observations that AAV mediates long-term and stable transgene expression in experimental animals. 9,15 AAV-ISN-T-Mediated Tumor Cell Apoptosis In Vivo. To explore the mechanism of tumor growth suppression mediated by oral administration of AAV-ISN-T, immunohistochemistry analysis of the subcutaneous tumor sections was performed and apoptosis was examined on a cellular basis.…”

“…The soluble TRAIL was detectable both in mouse serum and within liver cells. 9 This experiment provided important evidence that the gene product could be secreted into the general circulation and function as a soluble cytokine efficiently acting on metastatic tumor cells. Moreover, the AAV's advantages of natural defectiveness, lack of pathogenicity and stability to temperature and pH extremes, and solvents have led to its potential use for an oral administration strategy.…”

Preclinical study of rAAV2-sTRAIL: pharmaceutical efficacy, biodistribution and safety in animals

“…We previously established a recombinant AAV-mediated soluble TRAIL 95-281 gene expression for cancer therapy 25,26 and demonstrated that intraportal injection via the hepatic portal veins of rAAV-TRAIL 95-281 into the liver of an orthotopic-transplanted EL-4 mouse model mimicking liver cancer metastasis resulted in a significant suppression of tumor growth and prolonged survival, while normal hepatocyte toxicity is undetectable. Histological and biochemical analysis in tumor tissue and serum confirmed that TRAIL 95-281 in active trimeric form was stably expressed in relatively high level in hepatocytes and secreted into the serum.…”