Effective knock down of very high ABCG2 expression by a hammerhead ribozyme

BACKGROUND: Ribozymes are an effective tool to reduce the mRNA levels of specific target genes. Overexpression of the drug transport protein, ABCG2, has been associated with multidrug resistance in cancer cells. MATERIALS AND METHODS: An expression plasmid encoding a hammerhead ribozyme against the ABCG2 gene was stably transfected into multidrug-resistant MCF7/MX cells that express very high levels of the ABCG2 protein. The effect of the ribozyme was determined by quantitative real-time RT-PCR, Western blot and cytotoxicity assays. RESULTS: The ribozyme reduced ABCG2 mRNA levels to less than 10% of control values, which resulted in the concomitant reduction of ABCG2 protein levels and sensitization of the cells to mitoxantrone and methotrexate. CONCLUSION: The ribozyme used was highly effective in reducing the expression of its target gene, ABCG2, and was able to modulate the associated multidrug-resistant phenotype.     

RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model

Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.     

应用原位突变技术构建免疫球蛋白K链可变区基因克隆及表达载体

设计并合成了一对聚合酶链反应（ＰＣＲ）的通用引物，可以从小鼠杂交瘤的ｃＤＮＡ中扩增出免疫球蛋白（Ｉｇ）Ｋ链可变区（ＶＫ）基因。利用这一对引物和原位突变技术，在小鼠的一个ｇｅｎｏｍｉｃＶＫ基因的两端分别引入一个ＤＮＡ限制性内切酶的酶切位点，构建成一个ＩｇＶＫ基因的通用型克隆载体。并将ＶＫ基因片段同人ｋ链基因区基因片段重组，构建成人－鼠嵌合ｋ链基因表达载体。     

Transport of intracellularly active ribozymes to the cytoplasm

Ribozymes are RNA molecules with enzymatic activity which selectively bind and cleave specific target RNAs. To date, numerous studies directed toward the application of ribozymes in vivo have been performed and many successful experiments have been reported. However, to induce high-level activities of ribozymes in vivo, several factors must be considered. Here we report that the cytoplasmic localization of ribozymes is important for their intracellular activity in mammalian cells. Northern blot analysis revealed that a tRNA(Val) ribozyme, which can assume a cloverleaf structure similar to that of a native tRNA, is efficiently transported to the cytoplasm. In contrast, the tRNAiMet-driven ribozyme, which does not maintain the cloverleaf structure, remained predominantly in the nucleus. In correlation with the localization, the activity of the exported ribozyme was higher than that of the ribozyme retained in the nucleus. These results should provide insight into the design of ribozymes that have high-level activity in mammalian cells.     

Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors.

Chemically stabilized hammerhead ribozymes are nuclease-resistant, RNA-based oligonucleotides that selectively bind and cleave specific target RNAs. Due to their potential for specifically inhibiting gene expression, ribozymes are being investigated for therapeutic applications as well as for the elucidation of gene function. In particular, we have investigated ribozymes that target the mRNA of the vascular endothelial growth factor (VEGF) receptors because VEGF signaling is an important mediator of tumor angiogenesis and metastasis. Here we report pharmacodynamic studies testing anti-Flt-1 (VEGFR-1) and anti-KDR (VEGFR-2) ribozymes in animal models of solid tumor growth and metastasis. Ribozymes targeting either Flt-1 or KDR significantly inhibited primary tumor growth in a highly metastatic variant of Lewis lung carcinoma. However, only treatment with the anti-Flt-1 ribozyme resulted in a statistically significant and dose-dependent inhibition of lung metastasis in this model. The anti-Flt-1 ribozyme was then tested in a xenograft model of human metastatic colorectal cancer in which significant inhibition of liver metastasis was observed. Taken together, these data represent the first demonstration that synthetic ribozymes targeting VEGF receptor mRNA reduced the growth and metastasis of solid tumors in vivo.     

抑制VEGF表达的锤头状核酶系统

目的:建立和评估抑制血管内皮生长因子(VEGF)表达的锤头状核酶(hammerhead ribozyme)技术系统.方法:对VEGF121基因RNA序列进行二级结构分析,选择靶点;设计并构建针对VEGF的分泌肽RNA的可表达锤头状核酶(14)载体系统和VEGF-荧光色素酶融合基因报告质粒;通过试管内切割实验等方法评估核酶对于试管内转录得到的VEGF RNA切割特异性和效率;通过瞬时共转染实验和稳定转染实验评估核酶在细胞内对于VEGF RNA的切割效率.结果:设计和构建了对VEGF RNA二级结构水平上的暴露区( 8, 36和 71位点:核酶1,3和4)和非暴露区( 17位点:核酶2)4个锤头状核酶(14)的两套质粒;试管内切割检测表明,针对 8, 36和 71位点的核酶(1,3和4)可以有效地在试管内对VEGFRNA进行特异性切割,使其水平分别降至对照的61.7%,27.6%和44.8%(荧光色素酶活性)或66.3%,27.0%和30.0%(蛋白质水平);将核酶表达质粒与VEGF-LUC模板质粒瞬时共转染人SMMC-7721肝癌细胞,核酶1,3和4 VEGF-LUC水平分别降低到对照的81.4%,56.6%和69.1%;稳定表达针对VEGF的核酶1,3或4的SMMC-7721细胞株中,内源性VEGF RNA的水平降至对照水平的5%以下.对照未转染的、转染空载体的和转染了核酶2( 17)的SMMC-7721细胞.结论:分别针对VEGF 8, 36和 71位点锤头状核酶可有效地抑制VEGF的RNA水平.     

Maxizymes and small hairpin-type RNAs that are driven by a tRNA promoter specifically cleave a chimeric gene associated with leukemia in vitro and in vivo

Many leukemias result from chromosomal translocations that lead to the generation of chimeric oncoproteins. Chimeric gene for the promyelocytic leukemia-retinoic acid receptor alpha (PML-RAR alpha) is the hallmark of acute promyelocytic leukemia. Specific gene silencing of an oncogene is desirable for the treatment of these diseases. We have previously constructed an allosterically controllable ribozyme (designated maxizyme) targeted for bcr-abl chimeric mRNA, whose cleavage activity is induced only in the presence of a specific RNA sequence of interest. It has been demonstrated recently that RNA interference induced by short hairpin-type RNAs provides a powerful method for sequence-specific gene silencing. Here we report that DNA vector-based maxizymes and short hairpin-type RNAs driven by the promoter of a human gene for tRNA(Val) specifically inhibit the expression of the chimeric gene for PML-RAR alpha both in vitro and in vivo. Our findings confirm the potential utility of maxizymes and shRNAs as therapeutic agents.     

Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which has been implicated in inhibition of apoptosis and control of mitotic progression. The finding that survivin is overexpressed in most human tumors but absent in normal adult tissues has led to the proposal of survivin as a promising therapeutic target for anticancer therapies. We decided to evaluate the effects of a ribozyme-based strategy for survivin inhibition in androgen-independent human prostate cancer cells. We constructed a Moloney-based retroviral vector expressing a ribozyme targeting the 3' end of the CUA(110) triplet in survivin mRNA, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations obtained by infection with the retroviral vector of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were selected for the study. Ribozyme-expressing prostate cancer cells were characterized by a significant reduction of survivin expression compared to parental cells transduced with a control ribozyme; the cells became polyploid, underwent caspase-9-dependent apoptosis and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of prostate cancer cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted in athymic nude mice. These findings suggest that manipulation of the antiapoptotic survivin pathway may provide a novel approach for the treatment of androgen-independent prostate cancer.     

Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified,synthetic ribozyme

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.     

核酶对肝癌耐药细胞株BEL—7402／DOX耐药性的逆转作用

为逆转多药耐药ｍｄｒ－１基因产物Ｐ－ｇｐ蛋白所介导的肿瘤细胞对多种化疗药物的耐受性，作者设计合成了一种能切割ｍｄｒ－１ｍＲＮＡ第１９６密码子ＧＵＣ序列的锤头状核酶（Ｒｉｂｏｚｙｍｅ），并定向克隆于逆转录病毒载体。经ＰＡ３１７包装后，病毒上清感染ＢＥＬ－７４０２／ＤＯＸ细胞株。经Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交证实，ＰＡ３１７及转化的ＢＥＬ－７４０２／ＤＯＸ细胞中均有病毒的高表达，ＲＴ－ＰＣＲ结果表明，转化细胞中ｍｄｒ－１ｍＲＮＡ与未转化细胞相比明显减少甚至不能扩增出来，流式细胞技术检测发现转化细胞表面Ｐ－ｇｐ的表达与非转化细胞相比明显下降。ＭＴＴ法检测发现转化细胞对多种化疗药物重新产生较高的敏感性。结果提示，此Ｒｉｂｏｚｙｍｅ转化ＢＥＬ－７４０２／ＤＯＸ细胞后能有效抑制ｍｄｒ－１基因的表达，使已产生耐药的肝癌细胞的多药耐药表型发生逆转。     

c-erbB-2癌基因特异性核酶的体外切割作用

目的 构建ｃ－ｅｒｂＢ－２特异性核酶基因及其底物基因的体外转录载体并探讨其体外切割作用。方法 在计算机设计的核酶ＲＺＩ基因的３‘端加上新的限制性酶切位点ＥｃｏＲ Ｖ,先合成ＲＺＩ基因并将之与其底物分别克隆入体外转录载体ｐＧＥＭ３Ｚｆ（－）,经用ＥｃｏＲ Ｖ快速初选出含有ＲＥ１基因重组子并测序鉴定。体外转录物用＾３２Ｐ标记。体外切割作用在Ｍｇ＾＋＋的存在下,３７℃反应１小时,进行聚丙烯酰胺凝胶电泳,     

核酶在肿瘤基因治疗中应用的研究进展

核酶是一类具有催化活性的小分子RNA ,它能够特异性的与靶RNA分子结合后进行切割 ,从而能抑制目的基因的表达。设计合适的核酶分子能够阻断癌基因的异常表达 ,逆转肿瘤细胞MDR ,促进肿瘤细胞凋亡 ,有望成为肿瘤基因治疗的有效手段之一。     

切割MDR1 RNA的核酶(Ribozyme)对肝癌多药耐药细胞株BEL-7402/DOX化疗耐药性的逆转作用

为逆转肿瘤多药耐药基因(MDR1)产物P-gp蛋白所介导的肿瘤细胞对多种化疗药物的耐受性,设计合成了一种能切割MDR1 mRNA第196密码子GUC序列的锤头状核酶(Ribozlyme)并定向克隆于转录病毒载体pDOR-neo的BamH Ⅰ位点.经病毒包装细胞PA317包装后感染人肝癌多药耐药细胞株BEL-7402/DOX细胞,经G418筛选得到稳定的转化细胞株.Northem Blot杂交证实包装细胞PA317及转化的BEL-7402/DOX细胞中均有病毒的高表达,RT-PCR证实转化细胞中MDR1 mRNA与未转化细胞相比明显减少甚至不能扩增出来,流式细胞技术检测转化细胞P-gp的表达与非转化细胞的93.4～97.5%相比下降至8.2～14.6%.MTT法检测证实转化细胞对多种化疗药物重新产生较高的敏感性.结果表明,表达Ribozyme的逆转录病毒载体转化肝癌多药耐药细胞BEL-7402/DOX后能有效抑制MDR1的表达和翻译,使已产生耐药的肿瘤细胞的多药耐药表型发生逆转.     

表达抗-VEGF发夹状核酶腺病毒载体的构建及其对结肠癌生长的抑制作用

背景与目的：VEGF过表达提示结肠癌预后不良。本实验构建带有抗-VEGF发夹状核酶的复制缺陷型腺病毒，观察其对结肠癌VEGF表达及肿瘤生长的抑制作用。方法：将人工合成的抗-VEGF发夹状核酶DNA定向克隆至转移质粒pAdTrack—CMV多克隆位点，然后与病毒骨架质粒pAdEasy-1在细菌BJ5183中重组，在293a细胞中包装成完整病毒（命名为Ad-Rz）并复制扩增、纯化。RT-PCR检测病毒感染后抗-VEGF发夹状核酶在HT-29细胞中的表达、real-time PCR及ELISA检测其对VEGF mRNA及蛋白表达的抑制作用。观察Ad-Rz对HT-29细胞和裸鼠移植瘤生长的抑制作用，CD34标记检测肿瘤组织微血管密度（MVD）。结果：抗-VEGF发夹状核酶成功克隆至复制缺陷型腺病毒载体上。抗-VEGF发夹状核酶可在HT-29细胞中表达．Ad—Rz感染的HT-29细胞中VEGFmRNA的相对表达量大约为PBS组的45％（e〈0．05）。ELISA结果显示Ad—Rz感染后的细胞上清液中蛋白含量（A值）为0．455±0．35／百万细胞，低于PBS组（P〈0．05）。Ad—Rz对HT-29细胞的生长抑制无统计学意义（P〉0．05），但可显著抑制肿瘤组织内血管的形成（P〈0．05），Ad—Rz治疗的移植瘤增殖率低于PBS组，但差异无统计学意义（P〉0．05）。结论：腺病毒载体介导的抗-VEGF发夹状核酶可有效抑制HT-29细胞VEGF的表达及移植瘤组织内血管的生成。     

Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells.

T cells can be engineered to target tumor cells by transduction of tumor-specific chimeric receptors, consisting of an extracellular antigen-binding domain and an intracellular signaling domain. However, the peripheral blood of cancer patients frequently contains an increased number of T regulatory cells, which appear to inhibit immune reactivity. We have investigated the effects of T regulatory cells on chimeric T cells specific for the B-cell antigen CD19, as B-cell malignancies are attractive targets for chimeric T-cell therapy. When a CD19 single-chain Fv antibody was coupled to the CD3 zeta (zeta) chain, there was sharply reduced activity on exposure to T regulatory cells, measured by CD19+ target-induced proliferation and cytotoxicity. By contrast, expression in T cells of a chimeric receptor consisting of the intracellular portion of the CD28 molecule fused to the zeta-chain and CD19 single-chain Fv not only produced a higher proliferative response and an increased nuclear factor kappaB activation but also sustained these activities in the presence of T regulatory cells. These effects are seen whether the chimeric T cells are derived from normal donors or from patients with B-cell chronic lymphocytic leukemia, indicating the potential for clinical application in B cell malignancies.