Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase- and mitochondria-dependent pathways

Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.     

Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme.

Toward gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections in AIDS, Moloney murine leukemia virus-derived retroviral vectors were engineered to allow constitutive and tat-inducible expression of an HIV-1 5' leader sequence-specific ribozyme (Rz1). These vectors were used to infect the human CD4+ lymphocyte-derived MT4 cell line. The stable MT4 transformants expressing an HIV-1 RNA-specific ribozyme, under the control of the herpes simplex virus thymidine kinase (tk) promoter, were found to be somewhat resistant to HIV-1 infection as virus production was delayed. In cells allowing ribozyme expression under control of the simian virus 40 or cytomegalovirus promoter, the rate of HIV-1 multiplication was slightly decreased, and virus production was delayed by about 14 days. The highest level of resistance to HIV-1 infection was observed in MT4 cells transformed with a vector containing a fusion tk-TAR (trans activation-responsive) promoter to allow ribozyme expression in a constitutive and tat-inducible manner; no HIV-1 production was observed 22 days after infection of these cells. These results indicate that retroviral vectors expressing HIV-1 RNA-specific ribozymes can be used to confer resistance to HIV-1 infection.     

细胞周期蛋白D1特异性核酶表达载体的构建及其活性

应用mfold程序对锤头状核酶(ribozyme,Rz)和大鼠细胞周期蛋白(cyclin)D1基因的二级结构进行分析,设计合成锤头状Rz基因,通过RT-PCR扩增获得大鼠细胞周期蛋白D1目的基因,将Rz基因和细胞周期蛋白D1基因分别克隆入载体pGEM-3Zf( )中,体外转录Rz基因和靶基因并进行切割实验;将Rz基因与逆转录病毒载体pLXSN重组得到Rz真核表达载体pLXSN-Rz,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆,用RT-PCR检测细胞周期蛋白D1基因的表达。结果显示:针对目的基因的832位点设计合成了Rz832,成功获得Rz832基因、细胞周期蛋白D1mRNA的体外转录载体pGEM3Zf-Rz832和pGEM3Zf-cD1,经体外转录出Rz832(105nt)及细胞周期蛋白D1mRNA(1079nt)。体外切割实验证实Rz832能够特异性切割细胞周期蛋白D1mRNA,产生1014nt和65nt的切割产物,切割效率为80%。所构建的pLXSN-Rz832经酶切电泳、PCR鉴定显示,插入的Rz832序列大小约为57bp,与预期结果相同,经测序证实Rz832序列正确。转染pLXSN-Rz832的肝星状细胞(hepaticstellatecells,HSCs)细胞周期蛋白D1mRNA的表达受到明显抑制,仅为对照组的42.22%(t=-193.443,P<0.01),结果表明:Rz832能够在体外特异性切割细胞周期蛋白D1mRNA、并在HSC-T6细胞内有效抑制细胞周期蛋白D1基因的表达。     

Reduction in severity of a herpes simplex virus type 1 murine infection by treatment with a ribozyme targeting the UL20 gene RNA

Hammerhead ribozymes were designed to target mRNA of several essential herpes simplex virus type 1 (HSV-1) genes. A ribozyme specific for the late gene U(L)20 was packaged in an adenovirus vector (Ad-U(L)20 Rz) and evaluated for its capacity to inhibit the viral replication of several HSV-1 strains, including that of the wild-type HSV-1 (17syn+ and KOS) and several acycloguanosine-resistant strains (PAAr5, tkLTRZ1, and ACGr4) in tissue culture. The Ad-U(L)20 Rz was also tested for its ability to block an HSV-1 infection, using the mouse footpad model. Mouse footpads were treated with either the Ad-U(L)20 Rz or an adenoviral vector expressing green fluorescent protein (Ad-GFP) and then infected immediately thereafter with 10(4) PFU of HSV-1 strain 17syn+. Ad-U(L)20 ribozyme treatment consistently led to a 90% rate of protection for mice from lethal HSV-1 infection, while the survival rate in the control groups was less than 45%. Consistent with this protective effect, treatment with the Ad-U(L)20 Rz reduced the viral DNA load in the feet, the dorsal root ganglia, and the spinal cord relative to that of the Ad-GFP-treated animals. This study suggests that ribozymes targeting essential genes of the late kinetic class may represent a new therapeutic strategy for inhibiting HSV infection.     

Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma

BACKGROUND: Despite attempts to develop efficient viral-based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation. METHODS: We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans-complementing replication-incompetent adenoviral vectors that resulted in tumor-restricted oncolysis. We combined an E1-deleted non-replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus, to allow full replication competence when tumor cells were co-infected with both vectors. RESULTS: A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans-complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co-localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans-complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans-complementing system of viruses alone, and long-term survival was only seen in the trans-complementing vector treatment group. Intraperitoneal administration of a pseudo-wild-type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans-complementing vectors resulted in only mild liver abnormalities. CONCLUSIONS: The trans-complementing vector approach using a combination of E1- and E4-deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication-competent vectors by requiring the presence of both vectors in a cell to achieve replication competence.     

双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用

为探讨双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用,观察双位点核酶对单一核酶体外剪切的增强作用,同时比较串联核酶和混合核酶的体外切割作用,构建了核酶Rz1,Rz3, Rz1和Rz3的串联核酶(Rz13)体外转录载体, 经体外转录后切割靶RNA. 结果表明:双位点核酶,无论是串联或混合核酶均可增强单一核酶的体外切割作用, 串联和混合核酶中的单一核酶可独立发挥作用;当串联和混合数目为2个时,两者的切割效率差别不大(P＞0.05).     

Construction of adenovirus for high level expression of small RNAs in mammalian cells

A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.     

Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA.

The structural motif formed between a hammerhead ribozyme and its substrate consists of three RNA double helices in which the sequence 5' to the XUY is termed helix I and the sequence 3' to the XUY helix III. Two hammerhead ribozymes targeted to the tat gene of HIV-1SF2 were designed to study target specificity and the potential effect of helix I mismatch on ribozyme efficacy both in vitro and in vivo. The first ribozyme (Rz1) targeted to the 5' splicing region of the tat gene was designed to cleave GUC*A. In HIV-1IIIB the A is changed to a G. The second ribozyme (Rz2) was targeted to the translational initiation region of the tat gene which is highly conserved among a variety of HIV-1 isolates, including both HIV-1SF2 and HIV-1IIIB. In vitro cleavage studies demonstrated that Rz1 efficiency cleaved HIV-1SF2 substrate RNA, but not HIV-1IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1SF2 or HIV-1IIIB substrate with equal efficiency. Both ribozymes were cloned into the 3' untranslated region of the neomycin gene (neo) within the pSV2neo vector and transfected into the SupT1 human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1SF2 or HIV-1IIIB virus. While Rz1-expressing cells were significantly protected from HIV-1SF2 infection, they exhibited no protection when infected with HIV-1IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1SF2 and HIV-1IIIB in SupT1 cells. Expression of both ribozymes in these cells was demonstrated by Northern analysis. RT-PCR sequencing analysis confirmed the respective HIV-1 target sequence integrity. These data demonstrate the importance of the first base pair distal to the XUY within helix I of the hammerhead structure for both in vitro and in vivo ribozyme activities and imply that the effectiveness of the anti-HIV-1 ribozymes against appropriate target sequences is due to their catalytic activities rather than any antisense effect.     

Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes

We have developed a library of hairpin ribozyme genes that can be delivered and expressed in mammalian cells with the purpose of identifying genes involved in a specific phenotype. By applying the appropriate phenotypic selection criteria in tissue culture, we can enrich for ribozymes that knock down expression of an unknown gene or genes in a particular pathway. Once specific ribozymes are selected, their target binding sequence is used to identify and clone the target gene. We have applied this technology to identify a putative tumor suppressor gene that has been activated in HF cells, a nontransformed revertant of HeLa cells. Using soft agar growth as the selection criteria for gain of transformation, we have isolated ribozymes capable of triggering anchorage-independent growth. Isolation of one of these ribozymes, Rz 568, led to the identification and cloning of the human homologue of the Drosophila gene ppan, a gene involved in DNA replication, cell proliferation, and larval development. This novel human gene, PPAN, was verified as the biologically relevant target of Rz 568 by creating five additional "target validation" ribozymes directed against additional sites in the PPAN mRNA. Rz 568 and all of the target validation ribozymes reduced the level of PPAN mRNA in cells and promoted anchorage-independent growth. Exogenous expression of PPAN in HeLa and A549 tumor cells reduced their ability to grow in soft agar, underscoring its role in regulating anchorage-dependent growth. This study describes a novel method for gene discovery where the intracellular application of hairpin ribozyme libraries was used to identify a novel gene based solely on a phenotype.     

Central nervous system expression of IL-10 inhibits autoimmune encephalomyelitis

Multiple sclerosis, an inflammatory, demyelinating disease of the CNS currently lacks an effective therapy. We show here that CNS inflammation and clinical disease in experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis, could be prevented completely by a replication-defective adenovirus vector expressing the anti-inflammatory cytokine IL-10 (replication-deficient adenovirus expressing human IL-10), but only upon inoculation into the CNS where local infection and high IL-10 levels were achieved. High circulating levels of IL-10 produced by i. v. infection with replication-deficient adenovirus expressing human IL-10 was ineffective, although the immunological pathways for disease are initiated in the periphery in this disease model. In addition to this protective activity, intracranial injection of replication-deficient adenovirus expressing human IL-10 to mice with active disease blocked progression and accelerated disease remission. In a relapsing-remitting disease model, IL-10 gene transfer during remission prevented subsequent relapses. These data help explain the varying outcomes previously reported for systemic delivery of IL-10 in experimental autoimmune encephalomyelitis and show that, for optimum therapeutic activity, IL-10 must either access the CNS from the peripheral circulation or be delivered directly to it by strategies including the gene transfer described here.     

Comparative analysis of antitumor activity of CD40L, RANKL, and 4-1BBL in vivo following intratumoral administration of viral vectors or transduced dendritic cells

The tumor necrosis factor (TNF) family comprises a group of ligands that regulate cell proliferation, differentiation, activation, maturation and apoptosis through interaction with the corresponding TNF receptor family members. In this study, we have evaluated whether adenovirus-mediated intratumoral gene transfer of CD40L, RANKL, or 4-1BBL elicits an immune response to established murine MC38 and TS/A tumors. Intratumoral administration of the recombinant adenoviral vectors expressing CD40L, RANKL or 4-1BBL 7 days post-tumor cell inoculation resulted in significant inhibition of MC38 tumor growth for all three ligands when compared with control groups treated with either saline or control adenovirus. However, intratumoral injection of Ad-4-1BBL or Ad-CD40L resulted in a significantly stronger inhibition of TS/A tumor progression than did Ad-RANKL treatment. We also demonstrated that intratumoral administration of dendritic cells (DC) transduced with adenoviral vectors encoding the TNF-related ligands resulted in a significant inhibition of MC38 tumor growth as compared with control groups treated with Ad-LacZ-transduced DC or saline-treated DC. In addition, DC overexpressing CD40L secreted considerably more IL-12 and expressed higher levels of the co-stimulatory molecules, CD80, CD86 and CD40, than did DC overexpressing LacZ, 4-1BBL or RANKL. We have also demonstrated that DC/CD40L, DC/4-1BBL, and DC/RANKL survived significantly longer than control DC or DC infected with the LacZ vector. Taken together, these results demonstrate that adenoviral gene transfer of CD40L, RANKL or 4-1BBL elicit a significant antitumor effect in two different tumor models, with CD40L gene transfer inducing the strongest antitumor effect.     

Activated H-ras transforms rat intestinal epithelial cells with expression of alpha-TGF

We describe malignant transformation of cultured rat intestinal epithelial cells (IEC-18) by transfection with the activated human H-ras gene cloned from the EJ bladder carcinoma. Transformed cells showed a marked morphological change, expressed high levels of the transfected H-ras gene, were able to grow in agar and expressed antigenic markers identical with parental IEC-18 cells. When injected into syngeneic rats these cells formed rapidly growing tumors expressing the same intestinal-specific antigenic markers as the injected cells. Parallel to the high expression of H-ras mRNA in the transformants we document overexpression of rat alpha-TGF mRNA.     

可诱导肿瘤靶向性IFN-α2a重组腺病毒的构建及其表达

腺病毒载体广泛应用于恶性肿瘤的靶向性基因治疗的研究,但这些肿瘤靶向载体缺乏可控性,其疗效和安全性受到很大的影响,因此开发新型可诱导的生物肿瘤靶向载体是当今抗肿瘤靶向药物研究的当务之急。该研究构建了可诱导肿瘤靶向性IFN-α2a重组腺病毒。重组腺病毒能够有效感染人肝癌细胞株HepG2等多种肿瘤细胞株,RT-PCR和Western blot结果表明肿瘤细胞能高效表达IFN-α2a,而其非肿瘤细胞株L02几乎没有表达。诱导试验表明重组腺病毒Ad MT-Ⅱ-hTERT/IFN-α2a能被ZnSO4诱导表达。可诱导肿瘤靶向性重组腺病毒Ad MT-Ⅱ-hTERT/IFN-α2a成功构建为下一步体内外抑癌实验研究打下了基础。     

Feline immunodeficiency virus vectors. Gene transfer to mouse retina following intravitreal injection

Background

Transduction of the murine retinal pigmented epithelium (RPE) with adenovirus vectors requires technically difficult and invasive subretinal injections. This study tested the hypothesis that recombinant vectors based on feline immunodeficiency virus (FIV) could access the retina following intravitreal injection.

Methods

FIV vectors expressing E. coli β‐galactosidase (FIVβgal) were injected alone, or in combination with adenovirus vectors expressing eGFP, into the vitreous of normal mice and eyes evaluated for transgene expression. In further studies, the utility of FIV‐mediated gene transfer to correct lysosomal storage defects in the anterior and posterior chambers of eyes was tested using recombinant FIV vectors expressing β‐glucuronidase. FIVβgluc vectors were injected into β‐glucuronidase‐deficient mice, an animal model of mucopolysacharridoses type VII.

Results

The results of this study show that similar to adenovirus, both corneal endothelium and cells of the iris could be transduced following intravitreal injection of FIVβgal. However, in contrast to adenovirus, intravitreal injection of FIVβgal also resulted in transduction of the RPE. Immunohistochemistry following an intravitreal injection of an AdeGFP (adenovirus expressing green fluorescent protein) and FIVβgal mixture confirmed that both viruses mediated transduction of corneal endothelium and cells of the iris, while only FIVβgal transduced cells in the retina. Using the β‐glucuronidase‐deficient mouse, the therapeutic efficacy of intravitreal injection of FIVβgluc (FIV expressing β‐glucuronidase) was tested. Intravitreal injection of FIVβgluc to the eyes of β‐glucuronidase‐deficient mice resulted in rapid reduction (within 2 weeks) of the lysosomal storage defect within the RPE, corneal endothelium, and the non‐pigmented epithelium of the ciliary process. Transgene expression and correction of the lysosomal storage defect remained for at least 12 weeks, the latest time point tested.

Conclusion

These studies demonstrate that intravitreal injection of FIV‐based vectors can mediate efficient and lasting transduction of cells in the cornea, iris, and retina. Copyright © 2002 John Wiley & Sons, Ltd.

     

Tissue-specific expression of an anti-ras ribozyme inhibits proliferation of human malignant melanoma cells.

In this study, we have compared the efficacy of a tissue-specific promoter (tyrosinase promoter) with a viral promoter to express anti-ras ribozyme RNA in human melanoma cells. The retroviral vector containing the tyrosinase promoter was superior in its ability to suppress the human melanoma phenotype in vitro as characterized by changes in growth, melanin synthesis, morphology and H-ras gene expression. These data support the use of tissue-specific expression of anti-oncogene ribozymes as a rational therapeutic strategy in human cancers.     

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

BACKGROUND: Ocular neovascular disorders, such as diabetic retinopathy and age-related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper-dependent adenovirus (HD-Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization. METHODS: We first analyzed efficiency and stability of intraretinal gene transfer following intravitreous injection in mice. A HD-Ad vector expressing green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (HD-Ad/GFP) was compared with a first-generation (E1/E3-deleted) Ad vector carrying an identical GFP expression cassette (FG-Ad/GFP). We also constructed HD-Ad vectors expressing a soluble form of the VEGF receptor (sFlt-1) in a constitutive (HD-Ad/sFlt-1) or doxycycline (dox)-inducible (HD-Ad/S-M2/sFlt-1) manner and tested their therapeutic efficacy upon intravitreous delivery in a rat model of oxygen-induced retinopathy (OIR). RESULTS: HD-Ad/GFP promoted long-lasting (up to 1 year) transgene expression in retinal Müller cells, in marked contrast with the short-term expression observed with FG-Ad/GFP. Intravitreous injection of HD-Ad vectors expressing sFlt-1 resulted in detectable levels of sFlt-1 and inhibited retinal neovascularization by more than 60% in a rat model of OIR. Notably, the therapeutic efficacy of the inducible vector HD-Ad/S-M2/sFlt-1 was strictly dox-dependent. CONCLUSIONS: HD-Ad vectors enable stable gene transfer and regulated expression of angiostatic factors following intravitreous injection and thus are attractive vehicles for the gene therapy of neovascular diseases of the retina.     

Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.