We have developed and validated a new tumor-targeting gene therapy strategy based upon the targeting and replacement of human telomerase reverse transcriptase (hTERT) RNA, using a trans-splicing ribozyme. By constructing novel adenoviral vectors harboring the hTERT-targeting trans-splicing ribozymes with the downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by the cytomegalovirus (CMV) promoter, we demonstrated that this viral system selectively marks tumor cells expressing hTERT or sensitizes tumor cells to prodrug treatments. We confirmed that Ad-Ribo-LacZ successfully and selectively delivered a ribozyme that performed a highly specific trans-splicing reaction into hTERT-expressing cancer cells, both in vitro and in a peritoneal carcinomatosis nude mouse model. We also determined that the hTERT-specific expression of the suicide gene in the Ad-Ribo-HSVtk, and treatment with the corresponding prodrug, reduced tumor progression with almost the same efficacy as the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and in nude mouse HT-29 xenografts. These observations provide the basis for a novel approach to cancer gene therapy, and demonstrate that trans-splicing ribozymes can be employed as targeting anti-cancer agents which recognize cancer-specific transcripts and reprogram them, thereby combating cancerous cells.
Purpose: Our previous studies suggested that human telomerase reverse transcriptase (hTERT) RNA-targeting trans-splicing ribozyme could be a useful tool for cancer gene therapy. Here, we investigated whether adenoviruses harboring this ribozyme can be systemically delivered to mice, and whether they selectively mark tumors expressing hTERT and sensitize them to ganciclovir treatments. Experimental Design: We constructed adenoviral vectors containing modified hTERT-targeting trans-splicing ribozyme with downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (AdRibo-HSVtk) driven by a cytomegalovirus promoter. The tumor-specific trans-splicing reaction and the tumor-killing effect of adenoviruses harboring ribozyme were investigated both in vitro and in vivo using mice with intrahepatic colon cancer metastasis via systemic administration. The safety of systemic administration of the viruses was also evaluated. Results: We showed that Ad-Ribo-LacZ, when injected i.v., performs a highly specific transsplicing reaction on hTERT mRNA and that it selectively marks tumors expressing hTERT in mice. More importantly, i.v. injection of Ad-Ribo-HSVtk plus ganciclovir significantly reduced tumor burden, with minimal liver toxicity, in mice with metastatic liver cancer, compared with the untreated group (P = 0.0009). Moreover, animals receiving Ad-Ribo-HSVtk showed improved survival compared with controls (P < 0.0001).Conclusions: This study shows that systemically delivered adenovirus harboring trans-splicing ribozyme can recognize cancer-specific transcripts and reprogram them to combat the cancer cells. Use of trans-splicing ribozymes seems to be a potentially useful gene therapy for cancer.Colorectal cancer is one of the most common cancers worldwide, both in terms of incidence and mortality (1). Despite progress in technologies for diagnosis and screening, liver metastasis occurs in 20% to 70% of patients with colorectal cancer and is the leading cause of death. Surgical resection or surgery coupled with systemic chemotherapy of liver metastasis is the treatment currently available for these patients (2). However, only 25% of liver metastases are amenable to surgery and f60% of those patients relapse (3). Therefore, there remains a continued need for a universally effective treatment for cancer and for new therapeutic approaches.Ribozymes are potential tools for gene therapy because of their ability to discriminate targets that differ by a single nucleotide, and splice and edit RNA transcripts (4, 5). The selfsplicing group I intron from Tetrahymena thermophila was shown to catalyze its own excision without the aid of proteins and also perform a trans-splicing reaction that specifically cleaves target RNAs, leading to revision of gene information (6 -8). In performing this trans-splicing reaction, group I ribozyme recognizes and binds a substrate RNA, cleaves the targeted RNA, and splices a 3 ¶ exon sequence that is initially attached to the 3 ¶ end of the ribozyme (9 -11). The transsplicing ribozyme has theref...
We previously showed that a trans-splicing ribozyme reprograms tumor-related genes at the mRNA level, resulting in the expression of therapeutic genes and that this approach can be efficiently employed to target specific molecules. Here, we show that trans-splicing ribozyme technology can be applied in molecular imaging of specific RNA expression in living animals. We exemplify this concept successfully by imaging mouse cytoskeleton-associated protein 2 (mCKAP2) expression in intrahepatic tumor nodules using systemically delivered adenovirus harboring mCKAP2-specific trans-splicing ribozyme.
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