Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector.

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.

Sponges against miR-19 and miR-155 reactivate the p53-Socs1 axis in hematopoietic cancers.

Normal cell proliferation is controlled by a balance between signals that promote or halt cell proliferation. Micro RNAs are emerging as key elements in providing fine signal balance in different physiological situations. Here we report that STAT5 signaling induces the miRNAs miR-19 and miR-155, which potentially antagonize the tumor suppressor axis composed by the STAT5 target gene SOCS1 (suppressor of cytokine signaling-1) and its downstream effector p53. MiRNA sponges against miR-19 or miR-155 inhibit the functions of these miRNAs and potentiate the induction of SOCS1 and p53 in mouse leukemia cells and in human myeloma cells. Adding a catalytic RNA motif of the hammerhead type within miRNA sponges against miR-155 leads to decreased miR-155 levels and increased their ability of inhibiting cell growth and cell migration in myeloma cells. The results indicate that antagonizing miRNA activity can reactivate tumor suppressor pathways downstream cytokine stimulation in tumor cells.

Cell cycle inhibition therapy that targets stathmin in in vitro and in vivo models of breast cancer.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that have a critical role in the regulation of mitosis. Stathmin is expressed at high levels in breast cancer and its overexpression is linked to disease progression. Although there is a large body of evidence to support a role for stathmin in breast cancer progression, the validity of stathmin as a viable therapeutic target for breast cancer has not been investigated. Here, we used a bicistronic adenoviral vector that co-expresses green fluorescent protein and a ribozyme that targets stathmin messenger RNA in preclinical breast cancer models with different estrogen receptor (ER) status. We examined the effects of anti-stathmin ribozyme on the malignant phenotype of breast cancer cells in vitro and in xenograft models in vivo both as a single agent and in combination with chemotherapeutic agents. Adenovirus-mediated gene transfer of anti-stathmin ribozyme resulted in a dose-dependent inhibition of proliferation and clonogenicity associated with a G2/M arrest and increase in apoptosis in both ER-positive and ER-negative breast cancer cell lines. This inhibition was markedly enhanced when stathmin-inhibited breast cancer cells were exposed to low concentrations of taxol, which resulted in virtually complete loss of the malignant phenotype. Interestingly, breast cancer xenografts treated with low doses of anti-stathmin therapy and taxol showed regression in a majority of tumors, while some tumors stopped growing completely. In contrast, combination of anti-stathmin ribozyme and adriamycin resulted in only a modest inhibition of growth in vitro and in breast cancer xenografts in vivo. Although inhibition of tumor growth was observed in both the combination treatment groups compared with groups treated with single agent alone, combination of anti-stathmin therapy and taxol had a more profound inhibition of tumorigenicity, as both agents target the microtubule pathway. Clinically, these findings are highly relevant because taxol is one of the most active chemotherapeutic agents in breast cancer. These studies provide the proof-of-principle that stathmin provides an attractive molecular target, which could serve as a primary focus of novel approaches to breast cancer.

Ribozyme to TGF-beta1 mRNA abrogates immunosuppressive effects of human colorectal adenocarcinoma HRT-18 cells in vitro and in vivo.

Transforming growth factor-beta1 (TGF-beta1) is overexpressed in a variety of malignant epithelial tumors and was suggested to be a marker of colorectal cancer. Moreover, there is growing evidence that TGF-beta1 contributes to tumor progression by regulating tumor cell proliferation and differentiation, inducing a favorable tumor microenvironment, promoting migration and invasion, and suppressing macrophage cytotoxicity. Therefore, we stably transfected an anti-TGF-beta1 hammerhead ribozyme into the human colorectal adenocarcinoma cell line HRT-18. Expression of this ribozyme resulted in significant inhibition of TGF-beta1 expression on mRNA and protein level. This was associated with an enhanced tumor cell differentiation and a reduced tumor growth in vivo. The capability of tumor cells to suppress ROI production of co-cultivated human macrophages was abrogated in transfectants. Taken together, inhibition of TGF-beta1 in colorectal carcinoma cells might be an interesting therapeutic tool leading to reduced tumor cell growth and increased macrophage cytotoxicity. Thus, a gene-therapeutic approach using anti-TGF-beta1 ribozyme in combination with established anti-tumor agents is of great promise.

[Construction of adenovirus expressing anti-vascular endothelial growth factor hairpin ribozyme and its inhibition of growth of colorectal cancer HT-29 cells].

BACKGROUND AND OBJECTIVE: Oversexpression of vascular endothelial growth factor (VEGF) is correlated to poor prognosis of colorectal cancer. This study was to construct replication deficient adenovirus carrying anti-VEGF hairpin ribozyme and investigate its inhibitory effects on VEGF expression and growth of colorectal cancer HT-29 cells. METHODS: Anti-VEGF hairpin ribozyme was cloned into the transfer vector pAdTrack-CMV, and recombined with adenoviral backbone vector pAdEasy-1 in BJ5183 bacteria. The recombinant plasmids were packaged and amplified in 293 cells (named Ad-Rz). The expression of anti-VEGF ribozyme in HT-29 cells was examined by RT-PCR. Inhibition of Ad-RZ on VEGF mRNA and protein expressions were detected by real-time PCR and ELISA, respectively. Inhibition of cell growth was measured by MTT assay. HT-29 cell xenografts were established in nude mice and the microvessel density (MVD) was determined by CD34 staining. RESULTS: The anti-VEGF ribozyme was successfully inserted into the adenoviral vector. Anti-VEGF ribozyme was effectively expressed in HT-29 cells. After infection by Ad-RZ, the relative expression of VEGF mRNA was decreased to about(45+/-0.01)% of that of PBS control group in HT-29 cells (P<0.05); the amount of VEGF protein in the supernatant of Ad-Rz treated cells [(OD=(0.46+/-0.35)/million cells] was lower than that of PBS treated cells [(OD=(0.80+/-0.35)/million cells] (P<0.05). Although Ad-Rz did not significantly inhibit cell growth of HT-29 cells (P>0.05), it significantly inhibited tumor angiogenesis of HT-29 cell xenografts in nude mice, compared with PBS group (P<0.05). The proliferation rate of xenografts treated by Ad-Rz was lower than that treated by PBS, but the difference was not significant (P>0.05). CONCLUSION: Anti-VEGF hairpin ribozyme mediated by adenovirus could inhibit the expression of VEGF and the tumor angiogenesis in colorectal cancer HT-29 cells.

Unexpected off-targeting effects of anti-huntingtin ribozymes and siRNA in vivo.

Gene transfer strategies to reduce levels of mutant huntingtin (mHtt) mRNA and protein by targeting human Htt have shown therapeutic promise in vivo. Previously, we have reported that a specific, adeno-associated viral vector (rAAV)-delivered short-hairpin RNA (siHUNT-2) targeting human Htt mRNA unexpectedly decreased levels of striatal-specific transcripts in both wild-type and R6/1 transgenic HD mice. The goal of this study was to determine whether the siHUNT-2-mediated effect was due to adverse effects of RNA interference (RNAi) expression in the brain. To this end, we designed two catalytically active hammerhead ribozymes directed against the same region of human Htt mRNA targeted by siHUNT-2 and delivered them to wild-type and R6/1 transgenic HD mice. After 10 weeks of continuous expression, these ribozymes, like siHUNT-2, negatively impacted the expression of a subset of genes in the striatum. This effect was independent of rAAV transduction and specific to the targeting of a unique sequence in human Htt mRNA. After consideration of the known potential RNAi-specific toxic mechanisms, only cleavage of an unintended RNA target can account for the data reported herein. Thus, long-term rAAV-mediated RNAi in the brain does not, in and of itself, negatively affect striatal gene expression. These findings have important implications in the development of therapeutic RNAi for the treatment of neurological disease.

Constructing the eukaryotic expression vector to study preliminarily the functions of hammerhead ribozyme targeting base excision repair gene HOGG1.

OBJECTIVE: Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair (BER) pathway. Human 8-oxoguanine DNA glycosylase 1 (HOGG1) is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer A549 cells would be investigated. METHODS: According to computer design, two specific restriction site BamH I and EcoR I were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1 (+). The positive recombinants were screened by ampicillin resistance, and plasmids were extracted from the positive recombinants and digested by BamH I and EcoR I , and then were analyzed by agarose gel electrophoresis and DNA sequencing. The recombinants were transiently transfected into A549 cells. The positive recombinants were identified by reverse transcription-polymerase chain reaction (RT-PCR) targeting to NEO gene, which was a neomycin resistance gene for selection of stable cell lines and only existed in vectors. The changes of HOGG1 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or single cell gel electrophoresis (SCGE) between untransfected and transfected cells. RESULTS: The recombinants containing the ribozyme gene were successfully selected by restriction endonuclease digestion and agarose gel electrophoresis, and were further proved by DNA automatic sequencing. A549 cells containing the recombinants were identified by RT-PCR, because NEO genes were amplified only in cells transfected successfully. The expression of HOGG1 mRNA in A549 transfected with ribozyme gene was 36% significantly less than in control cells (P < 0.05). MTT assay showed that the sensitivity of transfected cells to adriamycin were significantly increased in comparison with untransfected cells (P < 0.05). The comet assay showed that the extent of DNA damage induced by adriamycin was worse in transfected cells than unrtransfected cells (P < 0.05), but there was no time-dependent reaction correlation observed in cells. CONCLUSION: The eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA was constructed successfully, and effectively inhibited the expression of HOGG1 gene in lung cancer A549 cells, and increased the cellular sensitivity to adriamycin, and will help to study deeply the functions of base excision repair genes-HOGG1.

Inhibitory effect of anti-fas ribozyme on apoptosis of mouse T cells.

BACKGROUND & OBJECTIVE: Tumor cells can express FasL, and induce apoptosis of activated T cells expressing Fas, which is so called "Fas counterattack". This study was to investigate inhibitory effect of anti-Fas ribozyme on apoptosis of mouse T cells, and explore a new way to enhance antitumor ability of T cells. METHODS: A hammerhead ribozyme targeting Fas mRNA was synthesized, its in vitro cleavage reaction was performed. Anti-Fas ribozyme was transfected into mouse spleen T cells by electroperation (ribozyme-transfected group), empty control and mock-transfected groups were set up. Fas expression on T cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. After treatment of anti-Fas antibody (JO(2)), cell apoptosis was measured by flow cytometry, proliferation of T cells was measured by MTT assay. In vitro killing activity of cytotoxic T lymphocytes (CTLs) was detected by lactate dehydrogenase (LDH) releasing assay. RESULTS: Anti-Fas ribozyme cleaved Fas mRNA efficiently with a rate of 60%. mRNA level of Fas was significantly lower on ribozyme-transfected cells than on control and mock-transfected cells (0.4 vs. 1.1, and 1.0, P < 0.01)u its protein level was according to this result. After treatment of JO(2), cell apoptosis rate was significantly lower in ribozyme-transfected group than in the rest 2 groups (35% vs. 86%, and 87%, P < 0.01), but cell proliferation rate was significantly higher in ribozyme-transfected group than in the rest 2 groups (208% vs. 100%, and 98%, P < 0.01). In vitro killing activity of CTLs was significantly stronger in ribozyme-transfected group than in the rest 2 groups (67% vs. 32%, and 31%, P < 0.01). CONCLUSION: Anti-Fas ribozyme can remarkably suppress Fas expression on mouse activated spleen T cells, and protect T cells from Fas-mediated apoptosis, which may enhance antitumor ability of T cells.

Inhibition of HIV-1 replication by RNA targeted against the LTR region.

OBJECTIVE: The use of small RNA molecules able to effect gene inactivation has emerged as a powerful method of gene therapy. These small inhibitory RNAs are widely used for silencing malignant cellular and viral genes. We have assayed a series of inhibitory RNAs named catalytic antisense RNAs, consisting of a catalytic domain, hairpin or hammerhead ribozyme, and an antisense domain. The aim of the present study was to evaluate the effect of these inhibitory RNAs on HIV-1 replication. METHODS: A series of expression vectors has been constructed for the intracellular synthesis of inhibitory RNAs, differing in the promoter that drives their synthesis. These inhibitory RNAs were designed to act at two possible cleavage sites in the long terminal repeat (LTR) region and the TAR domain was chosen as a target for the antisense domain. We have evaluated the effects of different inhibitory RNAs in HIV replication via changes in p24 antigen levels. Mobility shift assays have been used to check the binding capacity of inhibitory RNAs. RESULTS: Catalytic antisense RNA designed to target the LTR region of HIV-1 inhibited viral replication in an eukaryotic cell environment by more than 90%. The conventional hairpin and hammerhead ribozymes, however, failed to inhibit viral replication. CONCLUSIONS: The data provide preliminary evidence of a new class of inhibitory RNAs that can be used to block HIV replication. The results clearly show the importance of the ex vivo antisense effect in the inhibition achieved. A good correlation was found between the in vitro binding efficiency of the inhibitor RNA to the HIV-1 LTR and the inhibition of viral replication.

RNA silencing of checkpoint regulators sensitizes p53-defective prostate cancer cells to chemotherapy while sparing normal cells.

p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G(2) checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.

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.

An intron promotes the anti-bcr-abl activities of a retrovirally expressed ribozyme in chronic myeloid leukemia cells.

Ribozymes have been developed to cleave the bcr-abl transcripts and thereby suppress transforming activities of chronic myelogenous leukemia (CML) cells. However, the intracellular efficacy of vector-dependent ribozymes usually depends in part on their expression cassettes, which may affect their intracellular trafficking and distribution. In order to test effects of an intron in pre-fusion ribozyme on the anti-bcr-abl activities in CML cells, retroviral vectors harboring ribozyme expression cassettes with (RzI) or without (Rz) an intron-encoding sequence were used to transduce K562 cells. In terms of both reduction of the target bcr-abl mRNA and suppression of colony formation in soft agar and xenograft growth on SCID mice, the anti-bcr-abl efficacy of the RzI fusion ribozyme was significantly superior to that of Rz. These results also correlate with more cytoplasmic accumulation of the RzI fusion ribozymes than that of the Rz. This study suggests activities of a RNA polymerase II-driven fusion ribozyme against its targeted spliced mRNA are improved by incorporating an intron in its pre-splicing transcript. Noticeably, the improvement is contributed in part by subcellular co-localization.

Construction and transfection of a ribozyme targeting human caspase-3.

Caspase-3 is a key executioner cysteine protease involved in programmed cell death or apoptosis. A ribozyme to human caspase-3 was designed, tested by in vitro cleavage, and transfected into a drug-resistant variant (DLKP-A5F) of a human lung carcinoma cell line (DLKP). By both stable and transient transfection, this ribozyme was shown to be effective at down-regulating human caspase-3 mRNA and protein levels.

Hammerhead ribozyme targeting connective tissue growth factor mRNA blocks transforming growth factor-beta mediated cell proliferation.

PURPOSE: Excessive scarring following trauma or surgery of cornea, conjunctiva or retina can greatly impair visual outcome. At present, no agents are clinically available that selectively reduce activity of genes that regulate fibrosis. Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues, including cornea and conjunctiva. In this study, hammerhead ribozymes targeting CTGF mRNA were synthesized, kinetic parameters were measured, and the effect on TGF-beta-mediated cell proliferation was measured in cultured human fibroblasts. METHODS: The mRNA sequence of human CTGF was scanned for potential hammerhead ribozyme cleavage sites, and predicted secondary folding structures around the sites were calculated. Synthetic 12mer ribozymes and 33mer oligonucleotide mRNA targets corresponding to two sites were synthesized, and kinetic constants calculated from Hanes-Wolff plots of in vitro cleavage reactions. The ribozyme with higher percentage cleavage and kinetic rate was cloned into an expression plasmid (pTR-UF21) and stably transfected into cultured human fibroblasts. An inactive ribozyme plasmid served as a negative control. The effects of the ribozyme on expression of TGF-beta-induced CTGF mRNA and protein levels were measured using ELISA and real-time TaqMan quantitative RT-PCR. Finally, the effect of the CTGF ribozyme on TGF-beta-mediated proliferation of fibroblasts was measured using a non-radioactive cell proliferation microtiter assay. RESULTS: Of the eight potential hammerhead ribozyme cleavage sites in human CTGF mRNA, two sites (CHR 745, and CHR 859) were identified with optimal secondary folding. CHR 859 cleaved 94% of the target mRNA, compared to 46% cleavage for CHR 745 after 16 hr of reaction. CHR 859 had a K(m) of 1.56 microM and a K(cat) of 2.97 min(-1), while CHR 745 had a K(m) of 7.80 microM and a K(cat) of 5.7 min(-1). The turnover numbers (K(cat)/K(m)) of CHR 859 and CHR 745 were 1.9 x 10(6) M min(-1) and 7.4 x 10(5) M min(-1), respectively, indicating CHR 859 is 2.6 times more efficient than CHR 745 in destroying CTGF mRNA. Stable transfection of CHR 859 into human fibroblasts reduced CTGF mRNA levels 55% and protein levels 72% compared to the inactive ribozyme control. Furthermore, TGF-beta-induced cell proliferation was reduced 90% in fibroblasts stably transfected with CHR 859 compared to control cell groups. CONCLUSIONS: The CHR 859 hammerhead ribozyme cleaved human CTGF mRNA with high kinetic efficiency in vitro, effectively reduced levels of CTGF mRNA and protein in cultured human fibroblasts, and blocked TGF-beta-induced cell proliferation without nonspecific toxicity. These data support the concept that CTGF mediates TGF-beta-induced cell proliferation, and imply that regulating CTGF expression with ribozymes may be effective in reducing ocular scarring.

[Influence of the inhibitor of c-Met on the growth and motility of hepatocellular carcinoma cells].

OBJECTIVES: To explore the influence of c-Met inhibitor by synthetic c-Met antisense oligonucleotide, constructive c-Met antisense plasmid and the complex plasmid of U1SnRNA/ ribozyme/anti-Met on the growth and metastasis of hepatocellular carcinoma cells. METHODS: Gene transfection was operated by Lipofectin on SF7721 cells. The difference of the cells before and after transfection was compared by MTT, growth curves and transwell test in vitro. In vivo, the cells before and after transfection were implanted subcutaneously into nude mice respectively to observe tumor growth and metastasis. RESULTS: C-Met antisense oligonucleotide could inhibit the growth of hepatocellular carcinoma SF7721 cells (t=3.58, P<0.05). After transfection, the expression of c-Met protein decreased. Growth curves showed that the cells after transfection proliferated more slowly, about 50% of control cells (F=4.87, P<0.05), and their motility and invasiveness decreased, compared with those before transfected. In vivo experiment, tumors originated from c-Met antisense oligonucleotide treated cells and the antisense/ribozyme/U1SnRNA treated cells grew more slowly (about 54.5% of those from the control cells), and the latent prolonged. After 35 days, the average weight of tumors in the two group nude mice were lighter than that in the control group nude mice (F=5.17, P<0.05). CONCLUSION: Inhibition of c-Met expression by c-Met antisense oligonucleotide and the complex of antisense/ribozyme/U1SnRNA can inhibit the growth and metastasis of SF7721 hepatocarcinoma cells in vitro and in vivo.

Down-regulation of c-Met inhibits growth in the liver of human colorectal carcinoma cells.

Overexpression of c-Met, the protein tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma. To examine the role of c-Met on in vitro and in vivo growth of human colon tumor cell lines, stable subclones of the high metastatic human colorectal carcinoma cell line, KM20, isolated from a Dukes' D patient, with reduced c-Met expression were obtained after transfection with a c-Met-specific targeting ribozyme. The subclones were only modestly reduced in c-Met expression because of c-Met playing an important role in cellular survival. However, a 60-90% reduction in basal c-Met autophosphorylation and kinase activity were observed. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates and soft-agar colony-forming abilities. The in vivo growth of these cells was examined at both the ectopic SQ site and the orthotopic site of metastatic growth, the liver. SQ growth was delayed significantly in the c-Met down-regulated clones compared with controls, with tumors growing on loss of the ribozyme construct. In contrast, tumor incidence was significantly reduced when the c-Met down-regulated cells were grown in the orthotopic liver site. Thus, c-Met activation may be important in metastatic growth of colon tumor cells in the liver. Collectively these data demonstrate that a small reduction in c-Met protein levels leads to profound biological effects, and potential c-Met inhibitors may be of therapeutic value in treatment of colon cancer.

Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences.

RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequence-specific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4(+) T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4(+) T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection = 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4(+) cells expressing the RNase P-associated U5 EGS (560) molecule.

An assay system to detect the selective advantage of anti-HIV ribozyme expressing CD4+ T-lymphocytes.

This study was undertaken to establish an assay system to detect the survival advantage of anti-HIV ribozyme expressing cells under the selective pressure of HIV infection. In a mixture with wild type cells the proportion of ribozyme expressing cells was increased up to 12-fold. As a mechanism of the selective advantage an inhibition of HIV induced apoptotic cell death could be demonstrated. Furthermore, a dose dependency of the antiviral ribozyme effects was observed.

Cytoplasmic expression of ribozyme in zebrafish using a T7 autogene system.

A cytoplasmic ribozyme expression system, based on codelivery of a ribozyme vector, a T7 autogene vector, and T7 RNA polymerase (RNAP), has been developed and used to generate a specific phenotype in zebrafish by targeting a no tail (ntl) mRNA. The expression of the no tail ribozyme sequence is under the control of a tandem of two promoters: The T7 promoter and an adenoviral va 1 (pol III) promoter. The coinjection of the ribozyme vector pT7vaRz, the T7 autogene vector pT7T7, and the T7 RNAP resulted in rapid synthesis of the ribozyme against the ntl mRNA in the cytoplasm of the injected zebrafish embryos, generating no tail phenotypes in up to 10-20% of the injected embryos. The phenotypic change rates have been found to be related to the concentrations of the plasmid vectors and T7 RNAP injected and to the ratios of the three injected components. This cytoplasmic ribozyme expression system may be useful for efficiently targeting other mRNA and for various biomedical applications. These potential applications may include rapid identification of biological functions of novel genes from zebrafish and humans based on partial gene sequence information and gene therapy of genetic and acquired diseases.