Abstract 3533: Systemic delivery of CTNNB1 Dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin
Abstract 3533: Systemic delivery of CTNNB1 Dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin
Generation of recombinant adenovirus using the Escherichia coli BJ5183 recombination system
Methods in molecular medicine, 2007
One of the most time-consuming steps in the generation of adenoviral vectors is the construction ... more One of the most time-consuming steps in the generation of adenoviral vectors is the construction of recombinant plasmids. This chapter describes a detailed method for the rapid construction of adenoviral vectors. The method described here uses homologous recombination machinery of Escherichia coli BJ5183 to construct plasmids used in generation of adenoviral vectors. With this method, no ligation steps are involved in generating the plasmids, and any region of the adenoviral genome can be easily modified. Briefly, the full-length adenoviral genome flanked by unique restriction enzyme sites is first cloned into a bacterial plasmid. Next, the region of the viral genome to be modified is subcloned into a bacterial shuttle plasmid, and the desired changes are introduced by molecular biology techniques. The modified viral DNA fragment is gel-purified and cotransformed with the full-length plasmid, linearized in the targeted region, into BJ5183 cells. Homologous recombination in E. coli g...
One of the most challenging aspects of lung cancer therapy is the rapid acquisition of multidrug-... more One of the most challenging aspects of lung cancer therapy is the rapid acquisition of multidrug-resistant (MDR) phenotype. One effective approach would be to identify and downregulate resistance-causing genes in tumors using small interfering RNAs (siRNAs) to increase the sensitivity of tumor cells to chemotherapeutic challenge. After identifying the overexpressed resistance-related antiapoptotic genes (survivin and bcl-2) in cisplatin-resistant cells, the siRNA sequences were designed and screened to select the most efficacious candidates. Modifications were introduced in them to minimize off-target effects. Subsequently, the combination of siRNA and cisplatin that gave the maximum synergy was identified in resistant cells. We then demonstrated that the combination treatment of the selected siRNAs and cisplatin encapsulated in CD44-targeting hyaluronic acid (HA)-based self-assembling nanosystems reversed the resistance to cisplatin and delayed the tumor growth significantly (growth inhibition increased from 30 to 60%) in cisplatin-resistant tumors. In addition, no abnormalities in body weights, liver enzyme levels or histopathology of liver/spleen tissues were observed in any of the treatment groups during the study period. Overall, we demonstrate that the combination of siRNA-mediated gene-silencing strategy with chemotherapeutic agents constitutes a valuable and safe approach for the treatment of MDR tumors.
While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral... more While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.
Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicit... more Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.
gene expression duration only persisted 12-24 hours and its magnitude attenuated when a second do... more gene expression duration only persisted 12-24 hours and its magnitude attenuated when a second dose was given 30 days after a first treatment, the levels and duration of IFN expression recovered when the interval was increased to 90 days. Gene expression inversely correlated with an inflammatory response to the adenovirus (mainly CD8 positive T-cells and macrophages) in the urothelium. When a second dose of rAd was administered 30 days after the first dose, a massive increase in T-cells was observed within twelve hours after treatment. However, when a second dose of rAd was administered 90 days after the first dose, the T-cell accumulation was attenuated and peaked at seven days, comparable to the immune response observed after a single dose of rAd.
cytotoxicity can be provided by use of semi replicating retroviruses at greatly improved levels c... more cytotoxicity can be provided by use of semi replicating retroviruses at greatly improved levels compared to replication defective vectors; moreover, a combination of suicide gene therapy delivered by semireplication competent vectors, with concomitant immune potentiation, can lead to highly significant survival advantages.
agent, which can be used in syngeneic-immunocompetent tumor models. Experiments assessing the ant... more agent, which can be used in syngeneic-immunocompetent tumor models. Experiments assessing the antitumor effects of this novel virus in syngeneic and xenograft models are underway.
JNCI Journal of the National Cancer Institute, 2007
We determined the cytolytic potential and selectivity of Seneca Valley Virus-001 (SVV-001), a new... more We determined the cytolytic potential and selectivity of Seneca Valley Virus-001 (SVV-001), a newly discovered native picornavirus, in neuroendocrine and pediatric tumor cell lines and normal cells. Suitability of the virus for intravenous delivery in humans was assessed by blood inactivation assays. Safety was evaluated in vivo using an immune-competent mouse model, and efficacy was evaluated in vivo in athymic mice bearing tumors derived from human small-cell lung cancer and retinoblastoma cell lines.
Abstract 3533: Systemic delivery of CTNNB1 Dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin
Abstract 3533: Systemic delivery of CTNNB1 Dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin
Generation of recombinant adenovirus using the Escherichia coli BJ5183 recombination system
Methods in molecular medicine, 2007
One of the most time-consuming steps in the generation of adenoviral vectors is the construction ... more One of the most time-consuming steps in the generation of adenoviral vectors is the construction of recombinant plasmids. This chapter describes a detailed method for the rapid construction of adenoviral vectors. The method described here uses homologous recombination machinery of Escherichia coli BJ5183 to construct plasmids used in generation of adenoviral vectors. With this method, no ligation steps are involved in generating the plasmids, and any region of the adenoviral genome can be easily modified. Briefly, the full-length adenoviral genome flanked by unique restriction enzyme sites is first cloned into a bacterial plasmid. Next, the region of the viral genome to be modified is subcloned into a bacterial shuttle plasmid, and the desired changes are introduced by molecular biology techniques. The modified viral DNA fragment is gel-purified and cotransformed with the full-length plasmid, linearized in the targeted region, into BJ5183 cells. Homologous recombination in E. coli g...
One of the most challenging aspects of lung cancer therapy is the rapid acquisition of multidrug-... more One of the most challenging aspects of lung cancer therapy is the rapid acquisition of multidrug-resistant (MDR) phenotype. One effective approach would be to identify and downregulate resistance-causing genes in tumors using small interfering RNAs (siRNAs) to increase the sensitivity of tumor cells to chemotherapeutic challenge. After identifying the overexpressed resistance-related antiapoptotic genes (survivin and bcl-2) in cisplatin-resistant cells, the siRNA sequences were designed and screened to select the most efficacious candidates. Modifications were introduced in them to minimize off-target effects. Subsequently, the combination of siRNA and cisplatin that gave the maximum synergy was identified in resistant cells. We then demonstrated that the combination treatment of the selected siRNAs and cisplatin encapsulated in CD44-targeting hyaluronic acid (HA)-based self-assembling nanosystems reversed the resistance to cisplatin and delayed the tumor growth significantly (growth inhibition increased from 30 to 60%) in cisplatin-resistant tumors. In addition, no abnormalities in body weights, liver enzyme levels or histopathology of liver/spleen tissues were observed in any of the treatment groups during the study period. Overall, we demonstrate that the combination of siRNA-mediated gene-silencing strategy with chemotherapeutic agents constitutes a valuable and safe approach for the treatment of MDR tumors.
While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral... more While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.
Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicit... more Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.
gene expression duration only persisted 12-24 hours and its magnitude attenuated when a second do... more gene expression duration only persisted 12-24 hours and its magnitude attenuated when a second dose was given 30 days after a first treatment, the levels and duration of IFN expression recovered when the interval was increased to 90 days. Gene expression inversely correlated with an inflammatory response to the adenovirus (mainly CD8 positive T-cells and macrophages) in the urothelium. When a second dose of rAd was administered 30 days after the first dose, a massive increase in T-cells was observed within twelve hours after treatment. However, when a second dose of rAd was administered 90 days after the first dose, the T-cell accumulation was attenuated and peaked at seven days, comparable to the immune response observed after a single dose of rAd.
cytotoxicity can be provided by use of semi replicating retroviruses at greatly improved levels c... more cytotoxicity can be provided by use of semi replicating retroviruses at greatly improved levels compared to replication defective vectors; moreover, a combination of suicide gene therapy delivered by semireplication competent vectors, with concomitant immune potentiation, can lead to highly significant survival advantages.
agent, which can be used in syngeneic-immunocompetent tumor models. Experiments assessing the ant... more agent, which can be used in syngeneic-immunocompetent tumor models. Experiments assessing the antitumor effects of this novel virus in syngeneic and xenograft models are underway.
JNCI Journal of the National Cancer Institute, 2007
We determined the cytolytic potential and selectivity of Seneca Valley Virus-001 (SVV-001), a new... more We determined the cytolytic potential and selectivity of Seneca Valley Virus-001 (SVV-001), a newly discovered native picornavirus, in neuroendocrine and pediatric tumor cell lines and normal cells. Suitability of the virus for intravenous delivery in humans was assessed by blood inactivation assays. Safety was evaluated in vivo using an immune-competent mouse model, and efficacy was evaluated in vivo in athymic mice bearing tumors derived from human small-cell lung cancer and retinoblastoma cell lines.
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Papers by shanthi ganesh