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

Abstract

Mutations in the KRAS gene, which persistently activate RAS function, are most frequently found in many types of human cancers. Here, we proposed and verified a new approach against cancers harboring the KRAS mutation with high cancer selectivity and efficient anti-cancer effects based on targeted RNA replacement. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically target and reprogram the mutant KRAS G12V transcript to induce therapeutic gene activity in cells. Adenoviral vectors containing the specific ribozymes with downstream suicide gene were constructed and then infection with the adenoviruses specifically downregulated KRAS G12V expression and killed KRAS G12V-harboring cancer cells additively upon prodrug treatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically in vivo. Importantly, intratumoral injection of the adenoviruses with pro-drug treatment specifically and significantly impeded the growth of xenografted tumors harboring KRAS G12V through a trans-splicing reaction with the target RNA. In contrast, xenografted tumors harboring wild-type KRAS were not affected by the adenoviruses. Therefore, RNA replacement with a mutant KRAS-targeting trans-splicing ribozyme is a potentially useful therapeutic strategy to combat tumors harboring KRAS mutation.

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

“…To solve this problem and improve the therapeutic potential of this approach, safer, more efficient, and endogenously and exogenously regulated trans ‐splicing ribozymes are required (Figure ). Improvement of specificity of intracellular trans ‐splicing reaction and no off‐target effects were reported through 5′‐end rapid amplification of cDNA ends (RACE) PCR analysis of the total trans ‐spliced products in the target RNA‐expressing cells by the modification of ribozyme to contain EGS upstream of IGS and the P10 helix sequence (Jung & Lee, ; Kim et al, ; Kim et al, ; Kwon et al, ). Moreover, recent studies have reported the development of trans ‐splicing ribozymes for which the activities can be precisely regulated using endogenous circumstances (Kim & Lee, ) or tissue‐specific miRNAs (Kim et al, ; Won et al, ).…”

“…Moreover, in contrast to ASO‐ and siRNA‐based approaches and genome editing approaches, trans ‐splicing ribozyme would not require any kinds of endogenous and/or exogenous proteins that could induce cellular toxicity and potential immunogenicity, and control of target binding length or post‐transcriptional regulation could increase the ribozyme specificity and thus minimize off‐target effects. Because of this RNA editing ability, trans ‐splicing ribozymes have played an important role in the fields of gene therapy (Ban et al, ; Byun, Lan, Long, & Sullenger, ; Carter et al, ; Jung, Kwon, & Lee, ; Kastanos, Hjiantoniou, & Phylactou, ; Kim, Kim, Yang, Jeong, & Lee, ; Kwon et al, ; Phylactou, Darrah, & Wood, ; Rogers, Vanoye, Sullenger, & George, ; Ryu, Kim, & Lee, ; Shin, Sullenger, & Lee, ; Song & Lee, ; Won & Lee, ) and molecular imaging (Hong et al, ; Kim et al, ; Kim et al, ; So, Gowrishankar, Hasegawa, Chung, & Rao, ). In this review, we will describe trans ‐splicing ribozymes, particularly the group I intron‐derived types, as well as gene therapy applications.…”

At the Intersection of Biomaterials and Gene Therapy: Progress in Non-viral Delivery of Nucleic Acids

“…In malignancies and pathological tissue induction, where multiple pathways are dysregulated, repairing a single gene may not be sufficiently effective and hence require expression of multiple therapeutic genes. Trans -splicing ribozymes with reprogramming capabilities have been developed that not only remove viral transcripts and tumor-related genes but also induce cell death leading to elimination of virus-infected and cancer cells (Won and Lee, 2012; Carter et al, 2014; Kim et al, 2017). Since reprogramming genes comes with a great risk of unconstrained expression and unintended gene removal, it is paramount to introduce elements that maintain a check on the activity of trans -splicing ribozymes.…”

From basic researches to new achievements in therapeutic strategies of KRAS-driven cancers

“…This specific trans-splicing reaction with the ability of discrimination target RNA from non-target one, even with a single nucleotide difference, makes it as an attractive novel treatment strategy for KRAS point mutations. Regarding KRAS -G12V mutation as one of the most prevalent point mutation, Tetra hymena group I intron-based trans-splicing ribozyme designed for selective cleavage of KRAS -G12V transcript 195 . An accurate and specific intracellular trans-splicing reaction of the designed ribozyme systems with the KRAS -G12V target RNA, leads to efficient reduction of transcript level.…”

“…Except that replacement of RNA, concurrent induction of suicide gene activity resulting in cytotoxicity and effective retardation of cancer cells harboring KRAS mutation 196 . Moreover, trans-splicing and therapeutic anti-cancer gene activity was selectively and efficiently induced only in KRAS -mutant cancer cells without targeting of cells expressing wild-type KRAS 195 . …”