Trans-splicing Ribozyme Research Articles

Targeted suicide gene therapy for liver cancer based on ribozyme-mediated RNA replacement through post-transcriptional regulation

Hepatocellular carcinoma (HCC) has high fatality rate and limited therapeutic options. Here, we propose a new anti-HCC approach with high cancer-selectivity and efficient anticancer effects, based on adenovirus-mediated Tetrahymena group I trans-splicing ribozymes specifically inducing targeted suicide gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To confer potent anti-HCC effects and minimize hepatotoxicity, we constructed post-transcriptionally enhanced ribozyme constructs coupled with splicing donor and acceptor site and woodchuck hepatitis virus post-transcriptional regulatory element under the control of microRNA-122a (miR-122a). Adenovirus encoding post-transcriptionally enhanced ribozyme improved trans-splicing reaction and decreased human TERT (hTERT) RNA level, efficiently and selectively retarding hTERT-positive liver cancers. Adenovirus encoding miR-122a-regulated ribozyme caused selective liver cancer cytotoxicity, the efficiency of which depended on ribozyme expression level relative to miR-122a level. Systemic administration of adenovirus encoding the post-transcriptionally enhanced and miR-regulated ribozyme caused efficient anti-cancer effects at a single dose of low titers and least hepatotoxicity in intrahepatic multifocal HCC mouse xenografts. Minimal liver toxicity, tissue distribution, and clearance pattern of the recombinant adenovirus were observed in normal animals administered either systemically or via the hepatic artery. Post-transcriptionally regulated RNA replacement strategy mediated by a cancer-specific ribozyme provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Small Animal PET Imaging of hTERT RNA-Targeted HSV1-tk Gene Expression with Trans-Splicing Ribozyme.

Background: Trans-splicing ribozymes (TSR) are useful anticancer agents targeting cancer-specific transcripts and replacing the RNA to induce anticancer gene expression specifically and selectively in cancer cells. Similar to other gene therapy methods, it is also important to evaluate the transgene expression for target specificity and ribozyme activity. Materials and Methods: In this study, the authors performed in vivo small animal positron emission tomography (PET) imaging and biodistribution assay to evaluate human telomerase reverse transcriptase (hTERT) RNA-targeting-specific TSR, which directs the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene selectively in hTERT-positive tumors through targeted RNA replacement of the hTERT transcript. Results: The hTERT RNA-targeted HSV1-tk expression with TSR was monitored by PET imaging with 124I labeled 2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil, which is one of the thymidine derivatives acting as substrates for HSV1-tk, in hTERT-positive tumor-bearing mice. Conclusions: Imaging of hTERT RNA-targeted HSV1-tk expression by TSR could be used in the development of advanced gene therapy using tumor-specific TSR.

Therapeutic applications of group I intron-based trans-splicing ribozymes.

Since the breakthrough discovery of catalytic RNAs (ribozymes) in the early 1980s, valuable ribozyme-based gene therapies have been developed for incurable diseases ranging from genetic disorders to viral infections and cancers. Ribozymes can be engineered and used to downregulate or repair pathogenic genes via RNA cleavage mediated by trans-cleaving ribozymes or repair and reprograming mediated by trans-splicing ribozymes, respectively. Uniquely, trans-splicing ribozymes can edit target RNAs via simultaneous destruction and repair (and/or reprograming) to yield the desired therapeutic RNAs, thus selectively inducing therapeutic gene activity in cells expressing the target RNAs. In contrast to traditional gene therapy approaches, such as simple addition of therapeutic transgenes or inhibition of disease-causing genes, the selective repair and/or reprograming abilities of trans-splicing ribozymes in target RNA-expressing cells facilitates the maintenance of endogenous spatial and temporal gene regulation and reduction of disease-associated transcript expression. In molecular imaging technologies, trans-splicing ribozymes can be used to reprogram specific RNAs in living cells and organisms by the 3'-tagging of reporter RNAs. The past two decades have seen progressive improvements in trans-splicing ribozymes and the successful application of these elements in gene therapy and molecular imaging approaches for various pathogenic conditions, such as genetic, infectious, and malignant disease. This review provides an overview of the current status of trans-splicing ribozyme therapeutics, focusing on Tetrahymena group I intron-based ribozymes, and their future prospects. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Group I Intron-Based Therapeutics Through Trans-Splicing Reaction.

In 1982, the Cech group discovered that an intron structure in an rRNA precursor of Tetrahymena thermophila is sufficient to complete splicing without assistance from proteins. This was the first moment that scientists recognized RNAs can have catalytic activities derived from their own unique three-dimensional structures and thus play more various roles in biological processes than thought before. Several additional catalytic RNAs, called ribozymes, were subsequently identified in nature followed by intense studies to reveal their mechanisms of action and to engineer them for use in fields such as molecular cell biology, therapeutics, imaging, etc. Naturally occurring RNA-targeting ribozymes can be broadly classified into two categories by their abilities: Self-cleavage and self-splicing. Since ribozymes use base-pairing to recognize cleavage sites, identification of the catalytic center of naturally occurring ribozymes enables to engineer from "self" to "trans" acting ones which has accelerated to design and use ribozyme as valuable tools in gene therapy fields. Especially, group I intron-based trans-splicing ribozyme has unique property to use as a gene therapeutic agent. It can destroy and simultaneously repair (and/or reprogram) target RNAs to yield the desired therapeutic RNAs, maintaining endogenous spatial and temporal gene regulation of target RNAs. There have been progressive improvements in trans-splicing ribozymes and successful applications of these elements in gene therapy and molecular imaging approaches for various pathogenic conditions. In this chapter, current status of trans-splicing ribozyme therapeutics, focusing on Tetrahymena group I intron-based ribozymes, and their future prospects will be discussed.

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

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 pro-drugtreatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically invivo. 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.

Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.

In vitro study of type I intron-mediated dual reporter gene imaging for carcinoembryonic antigen

Objective To develop a specific trans-splicing intron ribozyme type Ⅰ-mediated dual reporter gene system (Rib53-Fluc-tk) for targeting CEA. Methods The novel CEA-targeting trans-splicing ribozyme with the downstream reporter system (Rib53-Fluc-tk) was constructed by genetic engineering technology. The trans-splicing reaction product was evaluated using the 131I-5-iodo-2'-fluro-1-beta-D-arabinofuranosy-luracil (FIAU) cellular uptake rates and the bioluminescence. Two-sample t test, the analysis of variance and the least significant difference (LSD) t test was performed for data analysis. Results The sequence of Rib53-Fluc-tk was proved by gene-sequencing test. Human MCF-7 breast cancer cells showed a high ratio of firefly luciferase/renilla luciferase (0.64±0.10, n=4). A 520 bp band of product existed, which matched with the predicted size using RNA from cells transfected with Rib53-Fluc-tk in MCF-7. Signals were detected by bioluminescence in human embryonic kidney 293T cells co-transfected with Rib53-Fluc-tk and pCDNA3.1-CEA. The labelling rate of 131I-FIAU was (64.02±4.79)% (n=3). The radiochemical purity was (95.96±1.07)% (n=3), and the stability of the radiocompound remained high in human serum at least for 24 h. The uptake of 131I-FIAU in 293T cells transfected with Rib53-Fluc-tk was (0.31±0.01)% (n=4), while it increased with the incubation time in 293T cells co-transfected with pCDNA3.1-CEA and Rib53-Fluc-tk and reached (1.40±0.06)% at 4.5 h (F=1 007.29, t=136.34, both P<0.01). Conclusions A novel and specific reporter gene in the cellular level was established. Taking advantage of trans-splicing reaction of the ribozyme, it could improve the specificity of the reporter gene imaging. Key words: Genes, reporter; Arabinofuranosyluracil; Carcinoembryonic antigen; Bioluminescence imaging

Abstract 3547: Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model

Abstract Human telomerase reverse transcriptase (hTERT) RNA and p53 tumour suppressor were previously suggested as useful targets for cancer gene therapy. In this study, we investigated the effects of systemically delivered adenoviruses harboring trans-splicing ribozyme that could target hTERT and induce p53 transcript in targeted cancer cells in vivo using mice with Hep3B human hepatocellular carcinoma cells that metastasized to the liver. We observed that intravenous injection of this virus (0.5 X1011 Virus) produced efficient regression of Hep 3B tumor metastasized nodules. Next we performed in vitro study to know whether it has a synergistic effects with CDDP, we observed that CDDP (2.5 ug/ml) increased cytotoxic effects in vitro given with the virus (10 MOI), but in vivo study revealed that addition of CDDP or arsenic hexoxide (As4O6) did not improve the therapeutic efficacy of the virus treatment. Next we performed toxicity test with 4 doses (0.5 X1011, 1 X1011, 2 X1011, and 5 X1011, Virus), which revealed that there were no toxic death, but 2-4 fold increased in AST and ALT with no changes in bilirubin, BUN, Creatinine levels. In conclusion, this study demonstrates that the adenoviruses harboring trans-splicing ribozyme targeting hTERT and inducing p53 transcript represents a safe and promising cancer treatment for hepatocellular carcinoma. [This study was supported by a grant from the National R&amp;D Program for Cancer Control, Ministry for Health, Welfare &amp; Family Affairs, Republic of Korea. (0820050), and National Research Foundation of Korea (NRF) grant funded by the Korea government(MEST) (20120002631).] Keywords: Adenovirus, trans-splicing ribozyme, p53 tumour suppressor, Human Telomerase Reverse Transcriptase, cancer Citation Format: Won Sup Lee, Hye Lim Kang, Arulkumar Nagappan, Jeong Won Yun, GonSup Kim, Soon Chan Hong, Sang-Jin Lee, In-Hoo Kim. Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2015-3547

Targeted Regression of Hepatocellular Carcinoma by Cancer-Specific RNA Replacement through MicroRNA Regulation.

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.

139. Controllable Target-Specific Transgene Induction Using an Allosteric trans-Splicing Ribozyme

Gene therapeutic approaches are needed that can achieve inducible, adjustable, and well-controlled expression of therapeutic genes while simultaneously suppressing expression of disease-causing genes for maximization of their efficacy. In this study, we proposed a new approach to address one of the main challenges that current gene therapeutic techniques meet, which is based on group I intron ribozyme. To this end, we established an exogeneously and genetically controllable system through design of an allosteric ribozyme that comprises a Tetrahymena group I-based trans-splicing ribozyme as an active domain for RNA replacement, a small molecule-specific RNAaptamer as a sensor domain, and a communication module as an active transfer domain. This allosteric trans-splicing ribozyme can be used as a system that is able to specifically target, and thus reduce the level of, certain disease-specific RNAs, and simultaneously provides the ability to artificially control transgene expression through RNA replacement in therapeutic targets by using exogenous small molecules to activate the function of the ribozyme. The effectiveness of this approach was assessed by constructing various ribozymes in combination with a theophylline-binding aptamer to identify an allosteric ribozyme whose activity is controlled by theophylline both in vitro and in cell culture assays. Moreover, to confirm allosteric regulation of transgene expression, we constructed adenoviral vectors encoding the ribozymes and validated theophylline-dependent RNA replacement in target RNA-expressing cells. Results demonstrate that an allosteric trans-splicing ribozyme is an applicable RNA-based framework for engineering external ligand-controlled gene expression regulatory systems that exhibit adjustable regulation, design modularity (therefore, broad applicability), and target specificity. Such ribozyme could therefore be useful to monitor and/or modulate fate of any cells (such as cancer cells) using specific endogenous or exogenous cues.

24. Targeted Anticancer Approach Through microRNA-Regulated Tumor-Specific RNA Replacement

We have previously presented that trans-splicing ribozymes based on Tetrahymena group I intron could be potent anti-cancer devices through diminishing target RNA level and simultaneously triggering anti-cancer gene activity only in cancer cells expressing the target RNA. Particularly, hTERT-targeting ribozyme was shown to specifically and efficiently retard tumors in various xenograft carcinomatosis nude mouse models. However, concerns for the hTERT-targeting approach include potential side effects to proliferating hTERT+ normal cells. In this study, to improve cancer-specificity of the ribozyme, we developed cancer-specific ribozyme expression construct via microRNA regulation. To circumvent hepatocellular carcinoma (HCC) as target models, we created tissue-specific microRNA-regulated ribozyme system by incorporating perfect antisense sequence against normal hepatocyte-selective miRNA-122a that is down-regulated in HCC to the 3’-UTR region of ribozyme. Adenovirus encoding the modified hTERT-targeting ribozyme specifically and efficiently induced transgene and cytotoxicity in liver cancers through cancer-specific RNA replacement. Moreover, specificity of cancer regression was highly increased without compromising its anti-cancer efficacy in xenograft orthotopic HCC model. Furthermore, to assess the effect of this system in syngeneic model, we constructed mTERT-targeting ribozyme harboring miRNA-122a target sites. Adenoviral vector encoding the modified ribozyme selectively and efficiently regressed mTERT+ HCC with minimal liver toxicity due to least ribozyme expression in the normal livers in syngeneic orthotopic mouse model. Noticeably, cytotoxicity caused by the adenovirus induced specific immunogenic cancer cell death in the syngeneic model, thereby completely suppressing secondary tumor challenge in the mouse. Therefore, specifically targeted transgene induction through microRNA-regulated RNA replacement provides novel strategies for efficient and safe cancer gene therapy.

Low-dose cisplatin converts the tumor microenvironment into a permissive state for HSVtk-induced antitumor immunity in HPV16-related tonsillar carcinoma

An adenovirus harboring the HSV thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme that targets telomerase is cytotoxic to cancer cells because it inhibits DNA replication (Ad5mTR). Furthermore, it induces anti-tumor immunity by activating cytotoxic T cells. Because multiple chemotherapeutic agents also activate cytotoxic T-cell immunity during the direct killing process of tumor cells, we herein explored whether low-dose cisplatin could synergize with cytotoxic Ad5mTR to potentiate its therapeutic effect by boosting anti-tumor immunity in a murine HPV16-associated tonsillar carcinoma model. Tumor regression was enhanced when low-dose (1 mg/kg) cisplatin was added to suicide gene therapy using Ad5mTR. Meanwhile, 1 mg/kg cisplatin alone had no tumor-suppressive effects and did not result in any systemic toxicity. Thus, cisplatin along with Ad5mTR improved tumor clearance by increasing the number of E7-specific CD8+ T cells. Specifically, analysis of the tumors and lymph nodes supported improved immune clearance by increasing the number of E7-specific CD8+ T cells inside tumors (40%, P < 0.05) as a result of the combination of suicide gene and cisplatin therapy. These results suggest that a low dose of cisplatin potentiates CD8+ T-cell-mediated anti-tumor immunity, and its addition to the HSVtk-based adenovirus results in additional therapeutic benefits for HPV16-positive head and neck cancer patients.

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

Low oxygen conditions, termed hypoxia, can affect cell survivals. Cells may adapt to hypoxic conditions through hypoxia response elements (HRE) such as erythropoietin enhancer or phosphoglycerate kinase element. Hypoxic conditions usually appear in solid tumors, and can cause resistance to radiotherapy or chemotherapy. In this study, a genetic approach based upon Tetrahymena group I ribozyme was developed, which can address the challenges induced by a hypoxic microenvironment. To this end, human telomerase reverse transcriptase (hTERT) targeting trans-splicing ribozymes whose expression and activity were induced by HRE under hypoxia were constructed. Luciferase reporter assay showed induction of the transgene to increase due to the hypoxia-inducible ribozymes through a specific trans-splicing reaction in hTERT-expressing cells under hypoxic conditions. Increase in the transgene expression was mainly due to the increased trans-splicing reaction through a concurrent increase of the ribozyme expression level. Moreover, hypoxia-inducible ribozyme with herpes simplex virus thymidine kinase as the 3′exon effectively induced cell death when treated with ganciclovir under both hypoxic and normoxic conditions. These results indicated that the trans-splicing ribozyme could be a target-specific and efficacious anti-cancer tool to overcome resistance to radio- and chemotherapy under hypoxic conditions.

Conditional and target-specific transgene induction through RNA replacement using an allosteric trans-splicing ribozyme.

Gene therapeutic approaches are needed that can simultaneously induce the well-controlled expression of therapeutic genes and suppress the expression of disease-causing genes for maximization of their efficacy. To address this challenge, we designed an allosteric ribozyme that comprises a Tetrahymena group I-based trans-splicing ribozyme as an active domain for RNA replacement, a small molecule-specific RNA aptamer as a sensor domain, and a communication module as an active transfer domain. The effectiveness of this approach was assessed by constructing various ribozymes in combination with a theophylline-binding aptamer to identify an allosteric ribozyme, which is controlled by theophylline both in vitro and in cells. Moreover, we constructed adenoviral vectors encoding the ribozymes and validated allosteric regulation of trans-gene expression via theophylline-dependent RNA replacement in target RNA-expressing cells. Results demonstrate that an allosteric trans-splicing ribozyme is an applicable RNA-based framework for engineering external ligand-controlled gene expression regulatory systems that exhibit adjustable regulation, design modularity, and target specificity.

Trans-splicing with the group I intron ribozyme from Azoarcus

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNA(Ile) group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5' terminus. In contrast to the previously optimized 5'-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5' portion and the ribozyme 3' exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme.

Efficient targeting and tumor retardation effect of pancreatic adenocarcinoma up-regulated factor (PAUF)-specific RNA replacement in pancreatic cancer mouse model

The soluble protein pancreatic adenocarcinoma up-regulated factor (PAUF) plays an important role in pancreatic tumor progression and has begun to attract attention as a therapeutic target for pancreatic cancer. We herein present PAUF RNA-targeting gene therapy strategies with both targeting and therapeutic function using trans-splicing ribozyme (TSR) in pancreatic cancer. We developed adenoviral PAUF-targeting TSR (Rz) containing a PAUF-specific internal guide sequence (IGS) determined by library screening. This Rz harbors suicide gene, herpes simplex virus thymidine kinase (HSV-tk) or firefly luciferase (Luc) as a transgene for 3′ exon replacement of PAUF RNAs. Ad-Rz-TK, Rz harboring the HSV-tk, showed significant inhibition of tumor growth in vivo as well as PAUF-dependent cell death in vitro via a successful trans-splicing reaction. Selective induction of Rz-controlled transgene in PAUF-expressing pancreatic cancer was confirmed through noninvasive in vivo imaging; a luminescence signal from Rz harboring Luc (Ad-Rz-Luc) was detectable only in pancreatic tumor sites, not in normal mice. In addition, a [125I] FIAU signal reflecting thymidine kinase expression through SPECT and ex vivo biodistribution was co-localized with the tumor sites when we treated with Ad-Rz-TK in orthotopic xenograft model. Taken together, these results imply that PAUF-targeting TSR can contribute to successful targeted gene therapy for pancreatic cancer.

Adenovirus Expressing Both Thymidine Kinase and Soluble PD1 Enhances Antitumor Immunity by Strengthening CD8 T-cell Response

Adenoviruses harboring the herpes simplex virus thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme targeting human telomerase reverse transcriptase (hTERT-TR) show marked and specific antitumor activity. In addition to inducing tumor cell death by direct cytotoxicity, it is becoming clear that HSVtk also induces antitumor immunity. Programmed death ligand 1 (PD-L1) expressed on tumor cell surfaces mediates tumor-induced immunoresistance by inhibiting PD1-expressing tumor-infiltrating T cells. Here, we explored whether a soluble form of PD1 (sPD1-Ig), which blocks PD-L1, could synergize with TERT-TR-regulated HSVtk to enhance the adenoviral therapeutic efficacy by boosting antitumor immunity. Tumor antigen released by HSVtk-transduced tumors successfully primed tumor antigen-specific CD8 T cells via dendritic cells (DC). Regression of murine tumors was markedly enhanced when sPD1-Ig was incorporated into the adenovirus as compared with a single-module adenovirus expressing only HSVtk. This effect was abolished by CD8 T-cell depletion. Consistent with this, following adoptive transfer of tumor antigen-specific CD8 T cells into tumor-bearing Rag1(-/-) mice, dual-module adenovirus significantly enhanced CD8 T cell-mediated tumor rejection. In addition, secondary tumor challenge at a distal site was completely suppressed in mice treated with a dual-module adenovirus. These results suggest that a dual-targeting strategy to elicit both tumor antigen priming and tumor-induced immunoresistance enhances CD8 T cell-mediated antitumor immunity.

Cancer-Specific Induction of Adenoviral E1A Expression by Group I Intron-Based Trans-Splicing Ribozyme

In this study, we describe a novel approach to achieve replicative selectivity of conditionally replicative adenovirus that is based upon trans-splicing ribozyme-mediated replacement of cancer-specific RNAs. We developed a specific ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to induce adenoviral E1A gene expression selectively in cancer cells that express the RNA. Western blot analysis showed that the ribozyme highly selectively triggered E1A expression in hTERT-expressing cancer cells. RT-PCR and sequencing analysis indicated that the ribozyme-mediated E1A induction was caused via a high fidelity trans-splicing reaction with the targeted residue in the hTERTexpressing cells. Moreover, reporter activity under the control of an E1A-dependent E3 promoter was highly transactivated in hTERT-expressing cancer cells. Therefore, adenovirus containing the hTERT RNA-targeting transsplicing ribozyme would be a promising anticancer agent through selective replication in cancer cells and thus specific destruction of the infected cells.

Computational prediction of efficient splice sites for trans-splicing ribozymes

Group I introns have been engineered into trans-splicing ribozymes capable of replacing the 3'-terminal portion of an external mRNA with their own 3'-exon. Although this design makes trans-splicing ribozymes potentially useful for therapeutic application, their trans-splicing efficiency is usually too low for medical use. One factor that strongly influences trans-splicing efficiency is the position of the target splice site on the mRNA substrate. Viable splice sites are currently determined using a biochemical trans-tagging assay. Here, we propose a rapid and inexpensive alternative approach to identify efficient splice sites. This approach involves the computation of the binding free energies between ribozyme and mRNA substrate. We found that the computed binding free energies correlate well with the trans-splicing efficiency experimentally determined at 18 different splice sites on the mRNA of chloramphenicol acetyl transferase. In contrast, our results from the trans-tagging assay correlate less well with measured trans-splicing efficiency. The computed free energy components suggest that splice site efficiency depends on the following secondary structure rearrangements: hybridization of the ribozyme's internal guide sequence (IGS) with mRNA substrate (most important), unfolding of substrate proximal to the splice site, and release of the IGS from the 3'-exon (least important). The proposed computational approach can also be extended to fulfill additional design requirements of efficient trans-splicing ribozymes, such as the optimization of 3'-exon and extended guide sequences.