A Novel A33 Promoter–Based Conditionally Replicative Adenovirus Suppresses Tumor Growth and Eradicates Hepatic Metastases in Human Colon Cancer Models

Abstract

Purpose: A33 antigen is a membrane-bound protein expressed in intestinal epithelium that is overexpressed in 95% of primary and metastatic colorectal carcinomas but is absent in most epithelial tissues and tumor types. We hypothesized that A33 promoter might be useful in the design of a conditionally replicative adenovirus for the treatment of colorectal cancer (CRC). Experimental Design: We cloned an A33 promoter fragment (A33Pr) that extends from -105 to +307 bp. Using luciferase activity as a reporter gene, we showed that A33Pr was active in CRC cell lines. We next constructed a conditionally replicative adenovirus named AV22EL where E1A was placed under the control of A33Pr. The tumor-specific oncolytic effect of AV22EL was investigated both in vitro and in vivo. Results: AV22EL induced specific in vitro lysis of human CRC cell lines that expressed A33 and have negligible lytic capacity on cells that lacked or had minimal A33 expression, including normal human colonic cells. In vivo, a marked reduction of tumor growth and increased long-term survival rates were observed in nude mice xenografted with s.c. CRC tumors. Combination with 5-fluorouracil induced an additive effect in vitro with no toxic effects in vivo. Remarkably, AV22EL completely eliminated established hepatic metastases in >90% of mice and restored hepatic function according to biochemical parameters. Its systemic administration induced E1A expression only in the hepatic metastasis but not in normal organs. Conclusions: These data show that AV22EL is a stringently regulated and potent oncolytic agent for the treatment of CRC.Colorectal cancer (CRC) is the second most common cause of cancer mortality in Western countries and claimed >50,000 lives a year only in the United States. 4 Close to 70% of patients that are affected by colorectal carcinoma undergo surgical resection and 30% to 40% of them develop a recurrent disease (1). The liver is the most common site of metastatic CRC and complete resection of hepatic metastases is the only curative option; however, surgery can be done only in 20% of patients at the time of diagnosis and 5-year survival rates average 25% to 40% despite adjuvant chemotherapy (2). Among patients with metastatic colorectal carcinoma receiving as first-line chemotherapy 5-fluorouracil (5-FU) and new medications, such as irinotecan, Xeloda, oxaliplatin, Erbitux, and Avastin, the median time to progression is 8 to 9 months and their mean survival is 15 to 20.5 months (3 -6). It seems therefore that current CRC treatments are rather ineffective on advanced disease, showing the need for more effective and specific therapeutics to significantly increase patients' survival.Conditionally replicative oncolytic adenoviruses (CRAd) have shown promising applications in cancer gene therapy (7,8). One strategy to achieve specific elimination of the tumor mass, avoiding negative undesired effects in contiguous normal tissue, is the use of tumor-selective transcriptional regulation to control the essential early E1 genes, whic...

Transgenic Expression of miR-222 Disrupts Intestinal Epithelial Regeneration by Targeting Multiple Genes Including Frizzled-7

“…The A33 promoter was used to drive intestinal epithelial tissue-specific overexpression of the genomic miR-222 precursor, as reported by others (24,25). Transgenic founders on a pure C57BL/6J background were established by pronuclear injection at the University of Maryland, Baltimore transgenic animal core.…”

A Novel CDC25B Promoter–Based Oncolytic Adenovirus Inhibited Growth of Orthotopic Human Pancreatic Tumors in Different Preclinical Models

“…Total E4 copies per sample were normalized with the amount of DNA present in each sample and reported as E4 copies/ng of DNA (16). Detailed information is described in Supplementary Materials and Methods.…”

“…Five days later, cell viability was established with MTS as described (16). All assays were carried out in six different replicates.…”

Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

“…However, during a phase III clinical trial, the combination of dl1520 and chemotherapy treatment was suspended due to limited therapeutic efficacy [31]. Another strategy to develop cancer selective Ads is to restrict the expression of essential viral regulatory genes, such as E1a, using cancer selective promoters [7,32]. Oncolytic Ads with E1a controlled by cancer-selective promoters, such as OBP-301 (Telomelysin®) driven by human telomerase reverse transcriptase (hTERT) promoter [33] and CV706 driven by prostate-specific antigen (PSA) promoter [34], have progressed to human clinical trials.…”