Abstract
BackgroundWe have previously developed an oncolytic serotype 5 adenovirus (Ad5) with chromogranin-A (CgA) promoter-controlled E1A expression, Ad[CgA-E1A], with the intention to treat neuroendocrine tumors, including carcinoids. Since carcinoids tend to metastasize to the liver it is important to fully repress viral replication in hepatocytes to avoid adenovirus-related liver toxicity. Herein, we explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control.Methodology/Principal FindingsAd[CgA-E1A-miR122], where E1A expression is further controlled by six tandem repeats of the target sequence for the liver-specific miR122, was constructed and compared to Ad[CgA-E1A]. We observed E1A suppression and replication arrest of the miR122-detargeted adenovirus in normal hepatocytes, while the two viruses killed carcinoid cells to the same degree. Repeated intravenous injections of Ad[CgA-E1A] induced liver toxicity in mice while Ad[CgA-E1A-miR122] injections did not. Furthermore, a miR122-detargeted adenovirus with the wild-type E1A promoter showed reduced replication in hepatic cells compared to wild-type Ad5 but not to the same extent as the miR122-detargeted adenovirus with the neuroendocrine-selective CgA promoter.Conclusions/SignificanceA combination of transcriptional (promoter) and post-transcriptional (miRNA target) regulation to control virus replication may allow for the use of higher doses of adenovirus for efficient tumors treatment without liver toxicity.
Highlights
Virotherapy is an emerging approach to treat cancer
Liver-Specific Expression of miR122 miR122 is expressed in hepatocytes and it is the most abundant miRNA molecule expressed in the adult liver where it makes up 70% of all miRNAs [15]
The miR122 expression level was approximately 7-fold higher in human hepatocytes than in HuH7.5, while miR122 was not expressed in the hepatoma cell line HepG2 or in the nonhepatic neuroendocrine tumor cells lines BON, SH-SY-5Y, SK-N-BE(2), Kelly or in primary carcinoid cells (Supporting Figure S1). miRNA studies have shown that the strand used for recognition of target mRNA is the one, whose 59 end is less tightly paired [16]
Summary
Virotherapy is an emerging approach to treat cancer. It utilizes genetically engineered viruses for selective infection and killing of tumor cells while leaving normal cells relatively unharmed. Transcriptional targeting is mainly achieved by replacing an endogenous viral promoter sequence, e.g., the adenovirus E1A promoter, with a mammalian tumor- or tissue-specific promoter [1,2] This strategy restricts virus replication to target cells where the promoter is active. Et al were able to restrict replication of an oncolytic coxsackievirus (CVA21) by incorporation of miRT sequences recognized by a muscle-specific miRNA This reduced replication of the CVA21 virus in normal muscle tissue and resulted in reduced muscle toxicity without compromising the tumor cell-killing ability [8]. Two recent publications have described miR122-detargeting of the human serotype 5 adenovirus (Ad5) to reduce adenovirusinduced liver toxicity [9,10] Both publications use the wildtype E1A promoter to control E1A and demonstrated that incorporation of miR122 target sequences in the 39UTR of E1A gene reduces E1A expression in hepatic cells. We explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control
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