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.     

Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis.

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3' splice site (side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A.B. & Johansen, S.D. (2004) Eur. J. Biochem. 271, 1015-1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3' splice site, for application in group I ribozyme-mediated trans-splicing of RNA. The improved ribozyme catalyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginase mRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis.     

Expression of an RNase P ribozyme against the mRNA encoding human cytomegalovirus protease inhibits viral capsid protein processing and growth

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.     

Genetic manipulation of telomerase in HIV-specific CD8+ T cells: enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization

A large proportion of the CD8(+) T cell pool in persons chronically infected with HIV consists of cells that show features of replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8(+) T cells, particularly in the context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8(+) lymphocytes show both an enhanced and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-gamma and TNF-alpha in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28 expression, the signature change of replicative senescence in cell culture, was retarded in those CD8(+) T cell cultures that had high levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving force behind several aspects of CD8(+) T cell dysfunction associated with replicative senescence. We also demonstrate reduced accumulation of the p16(INK4a) and p21(WAF1) cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8(+) T cells. Given the key role of CD8(+) T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements associated with replicative senescence may lead to novel types of immunotherapy.     

Naturally occurring heterologous trans-splicing of adenovirus RNA with host cellular transcripts during infection

The impact of viral infection on normal host RNA processing remains largely unexplored. We postulated that the high abundance of virally derived nuclear RNA in infected cells could impact host cell RNA splicing and viability. To test for aberrant RNA splicing we examined trans-splicing following infection with the replication-competent adenovirus mutant d11520 that lacks E1B 55 kDa protein. Trans-splicing was observed between viral RNA and several cellular precursor mRNAs, including beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Using a tetracycline-inducible model system simulating viral trans-splicing activity we observed that overexpression of a trans-splicing RNA specifically inhibited cell proliferation. These results demonstrate that heterologous trans-splicing occurs naturally during adenovirus infection and suggest that trans-splicing may contribute to disruption of cell function.     

Ribozyme as an approach for growth suppression of human pancreatic cancer

Ribozymes (catalytic RNAs, RNA enzymes) are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential, and have potentially important implications for cancer gene therapy. Point mutations in the K-ras oncogene are found in approx 90% of human pancreatic carcinomas, and can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. In this study, we focused on in vitro manipulation of ribozyme targeting of the mutated K-ras oncogene in a human pancreatic carcinoma cell line. We evaluated the efficacy of an anti-K-ras hammerhead ribozyme targeted against GUU-mutated codon 12 of the K-ras gene in cultured pancreatic carcinoma cell lines. The anti-K-ras ribozyme significantly reduced cellular K-ras mRNA level (GUU-mutated codon 12) when the ribozyme was transfected into the Capan-1 pancreatic carcinoma cells. The ribozyme inhibited proliferation of the transfected Capan-1 cells. These results suggested that this ribozyme is capable of reversing the malignant phenotype in human pancreatic carcinoma cells.     

Adenovirus-mediated suicide gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter induced apoptosis of ovarian cancer cell line

Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats (TTAGGG)(n) to chromosomal ends, and it is known to play an important role in cellular immortalization. Telomerase is highly active in most tumor cells, yet not in normal cells. As such, it may have possible applications in cancer gene therapy. Telomerase consists of two essential components, telomerase RNA template (hTR) and catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. We here tested the possibility of the utilization of the hTERT promoter in targeted cancer gene therapy. We cloned the hTERT promoter in the replace of the CMV promoter and sub-cloned HSV-TK gene to be controlled by hTERT gene promoter in adenovirus shuttle plasmid. Then we constructed recombinant adenovirus Ad-hT-TK, and infected them into normal and human gynecological cancer cell lines. Through these experiments, we identified the selective tumor specific cell death by Ad-hT-TK. Furthermore, FACS analysis and TUNEL assay suggests that the reduced viability is mediated through the induction of apoptosis, indicating that this approach may be a useful method for suppressing cancer growth in targeted cancer gene therapy. These results show that Ad-hT-TK could be used for gynecological cancer gene therapy.     

人端粒酶逆转录酶核酶抑制端粒酶活性

为有效切割端粒酶逆转录酶mRNA以降低端粒酶活性 ,从而使肿瘤细胞生长变慢 ,凋亡增加。设计并合成了针对端粒酶逆转录酶mRNA的锤头状核酶基因 ,构建了该核酶基因的体外转录和真核表达质粒。检测了该核酶对端粒酶逆转录酶mRNA的体外切割效力。并将该核酶基因转染至肿瘤细胞中 ,检测其对肿瘤细胞端粒酶活性和生物学性状的影响。结果表明 ,该核酶在体外和细胞内均能有效切割端粒酶逆转录酶mRNA ;在细胞内能明显抑制端粒酶活性 ,使细胞生长变慢 ,倍增时间延长。因而 ,该核酶可望成为有效的端粒酶抑制剂 ,在抑制肿瘤生长中发挥作用     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.