Seung-Hee Hong

Antitumor Effects of Systemically Delivered Adenovirus Harboring Trans-Splicing Ribozyme in Intrahepatic Colon Cancer Mouse Model

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 (Ad-Ribo-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 trans-splicing 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.

In Vivo Anticancer Activity of Methotrexate-loaded Layered Double Hydroxide Nanoparticles

A methotrexate (MTX)-loaded layered double hydroxide (LDH) nanoparticle system was synthesized by intercalating MTX into the interlayer spaces of LDH. In vivo pharmacokinetic study demonstrated that the MTX-LDH hybrid had similar kinetic behaviors as free MTX, showing a rapid decline in the plasma MTX level, with characteristics of a biexponential function. However, the hybrid system remarkably suppressed tumor growth in human osteosarcoma-bearing mice compared to an equivalent amount of free MTX. Using MTX-LDH nanoparticles, a significantly high amount of MTX was delivered to target tumor tissue, whereas a low level was found in normal tissues. Moreover, LDH nanocarriers did not accumulate in any specific tissue nor cause acute toxicity up to the applied dose for the hybrid system. These results suggest that the MTX-LDH nanohybrid system has great potential as an anti-cancer drug with enhanced in vivo anti-tumor activity and bioavailability in target tumor tissue along with reduced side effects.

Gene transfer using liposome-complexed adenovirus seems to overcome limitations due to coxsackievirus and adenovirus receptor-deficiency of cancer cells, both in vitro and in vivo

Use of adenoviruses as vehicle for gene therapy requires that target cells express appropriate receptors such as coxsakievirus and adenovirus receptor (CAR). We show here that CAR-deficiency in cancer cells, that limits adenoviral gene delivery, can be overcome by using adenovirus complexed with the liposome, Ad-PEGPE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]. We first confirmed that CT-26 mouse colon cancer cells are deficient in CAR by RT-PCR, and then showed that CT-26 cells infected with Ad-GFP/PEGPE exhibited highly enhanced expression of green fluorescent protein (GFP), compared with those infected with Ad-GFP. GFP expression depends on the dose of liposome and adenovirus. Luciferase expression in livers treated with Ad-luc/PEGPE was about 1,000-fold less than those infected with Ad-luc. In a liver metastasis mouse tumor model developed by intrasplenic injection of CT-26 cells, luciferase expression following i.v. injection of Ad-luc/PEGPE was significantly higher in tumors than in adjacent non-neoplastic liver. Following systemic administration of Ad-GFP/PEGPE, GFP expression increased in tumors more than in adjacent liver while the reverse was true following administration of Ad-GFP. In the latter case, GFP expression was higher in liver than in tumors. This study demonstrates that systemic delivery of PEGPE-adenovirus complex is an effective tool of adenoviral delivery as it overcomes limitation due to CAR deficiency of target cells while reducing hepatic uptake and enhancing adenoviral gene expression in tumors.

Molecular imaging of endogenous mRNA expression in a mouse tumor model by adenovirus harboring trans-splicing ribozyme.

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.

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.

Involvement of mitogen-activated protein kinases and p21Waf1 in hydroxyurea-induced G1 arrest and senescence of McA-RH7777 rat hepatoma cell line

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21Waf1, p27Kip1 and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-β-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21Waf1 was increased, while p27Kip1 and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21Waf1 over-expression.

615. Feasibility of hTERT-targeting trans-splicing ribozyme in tumor-targeted gene therapy

Cancer gene therapy without toxicity is hampered by lack of efficient tumor-directed therapeutic gene expression system. In the present study, we describe a new tumor-targeted gene therapy tool that is based upon trans-splicing ribozymes. Trans-splicing ribozymes are RNA-based catalysts capable of performing both cleavage and ligation reaction in target RNA. Thus a chimeric mRNA can be produced and new gene activities triggered in cells dependent on the presence of the target RNA. We chose hTERT-targeting trans-splicing ribozyme for efficient tumor-directed expression of therapeutic gene by transcriptional targeting and RNA replacement because human telomerase reverse transcriptase (hTERT) is expressed at high levels in 90% of malignant tumors and tumor cell lines but is absent in normal postmitotic tissues. After developing a group I intron-based ribozyme targeting hTERT with high fidelity and specificity, we constructed novel adenoviral vectors containing hTERT-targeting trans-splicing ribozymes with downstream lacZ gene (Ad-ribolacZ) or herpes simplex virus thymidine kinase gene (Ad-ribotk). This hTERT-targeting trans-splicing ribozyme could simultaneously reduce hTERT expression and trigger expression of a new gene, such as lacZ or HSVtk, in cells expressing hTERT. To determine whether new gene activity could be selectively induced in hTERT expressing cells by trans-splicing ribozyme, we transfected Ad-ribolacZ into hTERT-positive and -negative cell lines. Then lacZ expression was analyzed by X-gal staining and β-galactosidase enzyme activity assays. We observed 4 to 9-fold increase of lacZ expression in hTERT-positive cells but not in h-TERT-negative ones. In contrast, induction of lacZ expression by adenovirus harboring lacZ gene driven by CMV promoter (Ad-CMVlacZ) could be observed in both hTERT-negative and-positive cells. Those results suggest that selective induction of transgene activity in hTERT-dependent manner by Ad-ribolacZ was mediated by correct targeting of hTERT with trans-splicing ribozyme in hTERT-positive cells. Given the observation that the trans-splicing ribozyme induced transgene expression selectively in target RNA-expressing cells, we determined whether the ribozyme would trigger selective cytotoxicity in hTERT-expressing cells. After transfecting Ad-ribotk into hTERT-positive and -negative cell lines, selective cytotoxicity was monitored by MTT assays. Ad-CMVtk was used as control. We could observe that Ad-ribotk revealed more than 50% reduction in survival rate of hTERT-positive cells, while not in hTERT-negative ones. In control, reductions of cell survival were observed regardless of hTERT expression. Those observation of highly selective cytotoxicity rendered by Ad-ribotk suggest that hTERT-targeting trans-splicing ribozyme may be one of powerful tools in tumor-targeted gene therapy.