Abstract
Hepatocellular carcinoma (HCC) has a high fatality rate and limited therapeutic options with side effects and low efficacy. Here, we proposed a new anti-HCC approach based on cancer-specific post-transcriptional targeting. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically induce therapeutic gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To circumvent side effects due to TERT expression in regenerating liver tissue, liver-specific microRNA-regulated ribozymes were constructed by incorporating complementary binding sites for the hepatocyte-selective microRNA-122a (miR-122a), which is down-regulated in HCC. The ribozyme activity in vivo was assessed in mouse models orthotopically implanted with HCC. Systemic administration of adenovirus encoding the developed ribozymes caused efficient anti-cancer effect and the least hepatotoxicity with regulation of ribozyme expression by miR-122a in both xenografted and syngeneic orthotopic murine model of multifocal HCC. Of note, the ribozyme induced local and systemic antitumor immunity, thereby completely suppressing secondary tumor challenge in the syngeneic mouse. The cancer specific trans-splicing ribozyme system, which mediates tissue-specific microRNA-regulated RNA replacement, provides a clinically relevant, safe, and efficient strategy for HCC treatment.
Highlights
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver in adults, generally followed by liver cirrhosis, and is caused by risk factors such as the hepatitis B and C virus, HIV, obesity, diabetes, alcohol intake, or environmental exposures
We developed a trans-splicing ribozyme targeting human telomerase reverse transcriptase RNA, and evidenced that the ribozyme could efficiently induce suicide gene activity through target-specific trans-splicing reaction in cells, and in tumor xenografts models when treated with pro-drugs[13,14,15,16]
We first evaluated the availability of miR-122a target sites (122aT) for the selective transgene regulation (Supplementary Fig. 1)
Summary
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver in adults, generally followed by liver cirrhosis, and is caused by risk factors such as the hepatitis B and C virus, HIV, obesity, diabetes, alcohol intake, or environmental exposures. Tetrahymena based trans-splicing ribozyme has been shown to target and cleave a substrate RNA and trans-ligate the 3′ exon of the ribozyme onto the downstream U nucleotide of the cleaved target RNA in cells[7,8] This trans-splicing ribozyme has been used for therapeutic applications, such as in the treatment of human genetic or malignant diseases[9,10,11,12]. We combined post-transcriptional regulation with an RNA replacement strategy by developing TERT-targeting trans-splicing ribozymes regulated by liver-specific miR-122a (Fig. 1) and evaluated the cellular and in vivo specificity and efficacy as a tool for targeted HCC gene therapy to address the cancer-selectivity challenge raised by the TERT-targeting approach. Viral vectors encoding the ribozymes were constructed and their target RNA specificity, transgene controllability, tissue selectivity, liver toxicity, and anti-cancer efficacy were analyzed in mouse models orthotopically implanted with xenografts and syngeneic HCC
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