Selective and efficient retardation of cancers expressing cytoskeleton‐associated protein 2 by targeted RNA replacement

Abstract

Human cytoskeleton-associated protein 2 (hCKAP2) is upregulated and highly expressed in various human malignances. hCKAP2 has microtubule-stabilizing characteristics and potentially regulates the dynamics and assembly of the mitotic spindle and chromosome segregation, indicating that hCKAP2 plays important functions during mitosis. In this study, we evaluated hCKAP2 as a plausible anticancer target through development and validation of a targeted cancer gene therapy strategy based on targeting and replacement of hCKAP2 RNA using a trans-splicing ribozyme. This targeted RNA replacement triggered transgene activity via accurate trans-splicing reaction selectively in human cancer cells expressing the hCKAP2 RNA and simultaneously reduced the expression level of the RNA in the cells. Adenoviral vector encoding the hCKAP2-specific transsplicing ribozyme selectively induced cytotoxicity in tumor cells expressing hCKAP2. Moreover, intratumoral injection of the virus produced selective and efficient regression of tumor that had been subcutaneously inoculated with hCKAP2-positive colon cancer cells in mice with minimal liver toxicity. Furthermore, orthotopically multifocal hCKAP2-positive hepatocarcinoma established in mice were efficiently regressed by systemic delivery of adenoviral vector encoding the specific ribozyme under the control of a liver-selective phosphoenolpyruvate carboxykinase promoter with least hepatotoxicity. The results indicate that hCKAP2 RNA is a promising target for anticancer approach based on trans-splicing ribozyme-mediated RNA replacement.Specificity and safety are one of the major concerns in cancer therapy. Gene targeting may represent an attractive approach to regress cancer cells without harming normal cells.1-4 Thus, many studies are now directed at identifying specific genes related to human cancers and at the development of efficient tools specifically targeting the identified genes for cancer therapy.Human cytoskeleton-associated protein 2 (hCKAP2), also known as tumor-associated microtubule-associated protein, is highly upregulated in various human maliganacies. 5-7Although its role in tumorigenesis still remains unknown, a series of recent studies demonstrated that CKAP2 plays critical functions in the process of cell proliferation, 8 especially during mitosis.9,10 CKAP2 has been proposed to be required for regulation of the dynamics and assembly of the mitotic spindle through stabilization of the microtubule 9-11 and, moreover, for the fidelity of the chromosome segregation process and fully sustained activity of the spindle assembly checkpoint.12 Hyperstabilization of microtubules due to overexpression of CKAP2 was reported to frequently induce aneuploidy leading to genomic stability and tumorigenesis. 11Taken together, CKAP2 could be a potential molecular target for cancer therapy.Recently, we demonstrated that Tetrahymena Group I intron-based trans-splicing ribozyme could be a potent anticancer agent through selective targeting and RNA replacement of transcripts dominantly expre...

Selective expression of transgene using hypoxia-inducible trans-splicing group I intron ribozyme

“…Moreover, a trans-splicing ribozyme that could selectively induce anti-viral activity in viral infected cells through the specific replacement of hepatitis C virus RNA has also been reported . Recently, we reported that the ribozyme selectively induced therapeutic genes in target RNA-expressing tumors both in cells and in vivo, thus effectively regressing the tumors, via RNA replacement through the trans-splicing reaction of cancer-specific RNA transcripts such as human telomerase reverse transcriptase (hTERT) Song et al, 2009), carcinoembryonic antigen (Jung and Lee, 2006), alpha-fetoprotein (Won and Lee, 2007), human cytoskeleton-associated protein 2 (Ban et al, 2011), and AIMP2-DX2 RNA (Won and Lee, 2012). In this study, a hypoxia-inducible trans-splicing ribozyme was developed, targeting the hTERT to overcome the therapeutic resistance observed in hypoxic tumors (Fig.…”

Therapeutic applications of group I intron‐based trans‐splicing ribozymes

“…Accordingly, single gene repair would not be sufficiently effective. To overcome this limitation, our group and others developed trans ‐splicing ribozymes to reprogram viral transcripts (Carter et al, ; Carter, Keith, Barde, Fraser, & Fraser, ; Ryu et al, ; Ryu & Lee, ) or tumor‐related genes (Ban et al, ; Hong et al, ; Jeong et al, ; Jung & Lee, ; Kim et al, ; Kim et al, ; Kwon et al, ; Song et al, ; Won & Lee, ; Won & Lee, ; Won & Lee, ) and induce cell death, thus clearing virus‐infected or cancer cells (Figure ). This reprogramming leads to therapeutic gene expression only in virus‐infected or cancer cells, as long as the substrate is expressed only in diseased cells (such as in virally infected cells), and the ribozyme displays no off‐target effects such as splicing on unrelated cellular RNAs.…”

“…Won and Lee () developed an adenoviral hTERT‐targeting trans ‐splicing ribozyme in which the 3′‐exon had been exchanged with E1A, thus potentially converting a replication‐incompetent adenoviral vector to replication‐competent virus in hTERT‐expressing cells via a precise trans ‐splicing reaction. Trans ‐splicing ribozymes specific for carcinoembryonic antigen (CEA; Jung & Lee, ), cytoskeleton‐associated protein 2 (CKAP2; Ban et al, ; Kim et al, ), alpha‐fetoprotein (AFP; Won & Lee, ), and pancreatic adenocarcinoma upregulated factor (PAUF) RNA (Kim et al, ), all of which are over‐expressed in various cancers, have also been developed, and the abilities of these constructs to induce therapeutic transgene expression through target RNA reprograming have been specifically validated in target RNA‐expressing cells.…”

Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement

“…[26][27][28][29][30][31] Moreover, we designed ribozymes that can specifically transfer the therapeutic gene into target RNA-expressing cancer cells, resulting in effective retardation of tumors in vivo through RNA replacement by a trans-splicing reaction with cancer-specific RNA, such as human telomerase reverse transcriptase RNA or human cytoskeleton-associated protein 2 RNA. [32][33][34][35][36] In this study, we developed trans-splicing ribozymes that can specifically target the KRAS G12V transcript as a new targeting agent specifically against cancers harboring KRAS mutation. The ribozyme was designed to selectively splice and reprogram KRAS G12V transcripts, but not wild-type KRAS, inducing the therapeutic transgene specifically in cancer cells harboring the KRAS G12V mutation both in cells and in vivo.…”