- Email: [email protected]
289. Non-Viral siRNA Delivery for P-gp Downregulation in Breast Cancer Cell Therapy
CANCER - TARGETED GENE THERAPY I processes and timeline for product introduction into the clinic. Finally, the regulatory and clinical aspects are summarized for one further designer T cell application in prostate cancer with designer T cell engraftment that entered clinical trial in 2008. The information presented may be of value to investigators who are interested in the translation of laboratory discoveries to human use.
288. Feasibility of Non-Invasive Imaging To Detect Recombinant Adeno-Associated Virus 2-Mediated Long-Term Gene Expression for Cancer Gene Therapy
Ji Yun Kim,1 HanSaem Lee,1 Won Il Lee,1 Min Ji Ko,1 Dae Heak Moon,2 Heuiran Lee.1 1 Departments of Microbiology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of; 2Departments of Molecular Medicine, University of Ulsan College of Medicine, Seoul, Korea, Republic of. A recombinant adeno-associated virus (rAAV) is currently known to be as a promising cancer gene therapy vector owing to its longterm gene expression and non-pathogenicity. Herpes simplex virus 1-thymidine kinase (HSV-TK) gene delivered by rAAV can induce preferential cytotoxicity of human cancer cells following ganciclovir (GCV) treatment. Moreover, cells expressing HSV-TK can be monitored by non-invasive micro-positron emission tomography (micro-PET) using 18F-FHBG. In this study, we aimed to evaluate whether rAAV2 vector-mediated mutant HSV-sc39TK expression, could induce anti-tumoral effects not only in vitro, but also in vivo animal model by micro-PET. To monitor human tumor growth in Balb/c nude mice through micro-PET, human cancer cell lines were infected by rAAV2-GFP or -sc39TK before subcutaneous injection. Micro-PET scans were performed after injection of 18F-FHBG once a week till 7 weeks with or without intraperitoneal GCV treatment. Tumor growth rates were dependent on initial cell numbers inoculated, and HeLa tumor growth was relatively faster than that of SK-Hep1. Imaging data showed that signals by 18F-FHBG were closely related with rAAV-mediated HSV-TK expression in tumors. Monitoring of tumor growth by imaging was possible for over 5 to 7 weeks after tumor cell implantation. Tumor size expressing sc39TK was sharply reduced following GCV treatment. Necrotic features were readily noticed in sc39TK/GCV-treated tumor. At the same time, there was the significant loss of 18F-FHBG uptake in tumors treated with GCV. The present study thus suggests that persistent anti-tumoral effect via rAAV2-sc39TK can be successfully monitored by using non-invasive micro-PET imaging, indicating that it is a promising modality for monitoring the long-term gene expression in tumors via rAAV2. Keywords: human cancer cells, recombinant adeno-associated virus, HSV1-TK, sc39TK, long term expression, micro-positron emission tomography. * Heuiran Lee: corresponding author; † JY Kim and HS Lee equally contributed in this study.
289. Non-Viral siRNA Delivery for P-gp Downregulation in Breast Cancer Cell Therapy
Meysam Abbasi,1 Vanessa Incani,3 Afsaneh Lavasanifar,3 Hasan Uludag.1,2,3 1 Biomedical Engineering, University of Alberta, Edmonton, AB, Canada; 2Chemical & Materials Engineering, University of Alberta, Edmonton, AB, Canada; 3Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada. Purpose: The aim of this study was to pursue an effective approach to non-viral siRNA delivery in breast cancer treatment. siRNA delivery by poly(L-Lysine) (PLL), stearic acid-modified PLL (PLLStA), polyethylenimine (PEI), oleic acid-modified PEI (PEI-OA) and LipofectamineTM 2000 was explored to suppress P-glycoprotein (P-gp) Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
expression, the main factor causing multi drug resistance (MDR) in chemotherapy of breast cancer. By suppressing P-gp expression, one can reduce the number of P-gp molecules on cell surfaces, reducing efflux of anticancer drugs mediated by the P-gp. Methods: The breast cancer cells MDA435/LCC6 MDR and their wild type (WT) phenotype were seeded in multiwell plates for a variety of studies. P-gp content was assessed by using FITC-labeled anti-human P-gp antibody. Gel electrophoresis was performed for assessment of (i) siRNA binding by carriers, (ii) siRNA complex dissociation upon incubation with heparin, and (iii) siRNA degradation in fresh rat serum. A flow cytometry assay for cellular uptake of doxorubicine (DOX) was used in parallel with a cytotoxicity assay (MTT) after cell treatment with either DOX or Paclitaxel (PTX). Flow cytometry was also used to assess siRNA uptake into cells after treating the cells with carrier/siRNA complexes (3.5:0.35 µg/mL carrier:siRNA). Finally, a specific siRNA sequence for P-gp down-regulation was delivered via the carriers and P-gp signal was assessed at different time points by flow cytometry. Results: Approximately 90% of the MDR cells was shown to contain P-gp compared to 1% of WT cells, in line with higher uptake of DOX into WT cells. MTT assay also showed that WT cells were more sensitive to DOX and PTX treatment especially at higher concentrations (>0.3µg/mL). siRNA uptake into the MDR and WT cells by PLL-StA showed considerably high siRNA uptake by both types of cells (60-70%) with no significant differences. Intracellular presence of siRNA was short (∼24 hours) irrespective of the carrier used. Using P-gp specific siRNA, Lipofectamine TM 2000 led to the highest down-regulation of P-gp (∼80%), while polymeric carriers yielded ∼40-55% P-gp downregulation. The polymers, however, were able to protect the siRNA against serum to a better extent than LipofecatmaineTM. Conclusions: The amount of siRNA uptake into cells non-viral carriers is considerably high, which led to significant down-regulation of P-gp. Although siRNA has a short half-life inside the cells, this was sufficient for P-gp inhibition. Future studies are planned to probe correlations between drug accumulation and P-gp inhibition, as well as identifying the most effective carrier for such a siRNA delivery.
290. Improved Gene Transfer into Renal Carcinoma Cells Using an Adenovirus Vector
Shuji Terao,1 Bishnu Acharya,1 Toru Suzuki,1 Katsuyuki Hamada,2 Hiroyuki Mizuguchi,3 Akinobu Gotoh.1 1 Laboratory of Cell and Gene Therapy, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; 2Department of Obstetrics and Gynecology, Ehime University School of Medicine, Toon, Japan; 3Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. Purpose: The transduction efficacy of adenovirus serotype 5 (Ad5) vector in human renal carcinoma cells is generally extremely low due to the non-expression of Coxsackie and adenoviral receptor (CAR), for which Ad5 has a high affinity. We investigated whether the histone deacetylase inhibitor, valproic acid (VPA), or fiber-modified Ad vector containing an RGD motif in the HI loop of the Ad fiber knob could increase the transduction efficiency of Ad5 in human renal carcinoma cells in vitro. Methods: We have tested human renal carcinoma cell lines Caki-1, ACHN, RCC4-VHL, and 786-O, and human bladder S113
288. Feasibility of Non-Invasive Imaging To Detect Recombinant Adeno-Associated Virus 2-Mediated Long-Term Gene Expression for Cancer Gene Therapy
Ji Yun Kim,1 HanSaem Lee,1 Won Il Lee,1 Min Ji Ko,1 Dae Heak Moon,2 Heuiran Lee.1 1 Departments of Microbiology, Research Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul, Korea, Republic of; 2Departments of Molecular Medicine, University of Ulsan College of Medicine, Seoul, Korea, Republic of. A recombinant adeno-associated virus (rAAV) is currently known to be as a promising cancer gene therapy vector owing to its longterm gene expression and non-pathogenicity. Herpes simplex virus 1-thymidine kinase (HSV-TK) gene delivered by rAAV can induce preferential cytotoxicity of human cancer cells following ganciclovir (GCV) treatment. Moreover, cells expressing HSV-TK can be monitored by non-invasive micro-positron emission tomography (micro-PET) using 18F-FHBG. In this study, we aimed to evaluate whether rAAV2 vector-mediated mutant HSV-sc39TK expression, could induce anti-tumoral effects not only in vitro, but also in vivo animal model by micro-PET. To monitor human tumor growth in Balb/c nude mice through micro-PET, human cancer cell lines were infected by rAAV2-GFP or -sc39TK before subcutaneous injection. Micro-PET scans were performed after injection of 18F-FHBG once a week till 7 weeks with or without intraperitoneal GCV treatment. Tumor growth rates were dependent on initial cell numbers inoculated, and HeLa tumor growth was relatively faster than that of SK-Hep1. Imaging data showed that signals by 18F-FHBG were closely related with rAAV-mediated HSV-TK expression in tumors. Monitoring of tumor growth by imaging was possible for over 5 to 7 weeks after tumor cell implantation. Tumor size expressing sc39TK was sharply reduced following GCV treatment. Necrotic features were readily noticed in sc39TK/GCV-treated tumor. At the same time, there was the significant loss of 18F-FHBG uptake in tumors treated with GCV. The present study thus suggests that persistent anti-tumoral effect via rAAV2-sc39TK can be successfully monitored by using non-invasive micro-PET imaging, indicating that it is a promising modality for monitoring the long-term gene expression in tumors via rAAV2. Keywords: human cancer cells, recombinant adeno-associated virus, HSV1-TK, sc39TK, long term expression, micro-positron emission tomography. * Heuiran Lee: corresponding author; † JY Kim and HS Lee equally contributed in this study.
289. Non-Viral siRNA Delivery for P-gp Downregulation in Breast Cancer Cell Therapy
Meysam Abbasi,1 Vanessa Incani,3 Afsaneh Lavasanifar,3 Hasan Uludag.1,2,3 1 Biomedical Engineering, University of Alberta, Edmonton, AB, Canada; 2Chemical & Materials Engineering, University of Alberta, Edmonton, AB, Canada; 3Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada. Purpose: The aim of this study was to pursue an effective approach to non-viral siRNA delivery in breast cancer treatment. siRNA delivery by poly(L-Lysine) (PLL), stearic acid-modified PLL (PLLStA), polyethylenimine (PEI), oleic acid-modified PEI (PEI-OA) and LipofectamineTM 2000 was explored to suppress P-glycoprotein (P-gp) Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
expression, the main factor causing multi drug resistance (MDR) in chemotherapy of breast cancer. By suppressing P-gp expression, one can reduce the number of P-gp molecules on cell surfaces, reducing efflux of anticancer drugs mediated by the P-gp. Methods: The breast cancer cells MDA435/LCC6 MDR and their wild type (WT) phenotype were seeded in multiwell plates for a variety of studies. P-gp content was assessed by using FITC-labeled anti-human P-gp antibody. Gel electrophoresis was performed for assessment of (i) siRNA binding by carriers, (ii) siRNA complex dissociation upon incubation with heparin, and (iii) siRNA degradation in fresh rat serum. A flow cytometry assay for cellular uptake of doxorubicine (DOX) was used in parallel with a cytotoxicity assay (MTT) after cell treatment with either DOX or Paclitaxel (PTX). Flow cytometry was also used to assess siRNA uptake into cells after treating the cells with carrier/siRNA complexes (3.5:0.35 µg/mL carrier:siRNA). Finally, a specific siRNA sequence for P-gp down-regulation was delivered via the carriers and P-gp signal was assessed at different time points by flow cytometry. Results: Approximately 90% of the MDR cells was shown to contain P-gp compared to 1% of WT cells, in line with higher uptake of DOX into WT cells. MTT assay also showed that WT cells were more sensitive to DOX and PTX treatment especially at higher concentrations (>0.3µg/mL). siRNA uptake into the MDR and WT cells by PLL-StA showed considerably high siRNA uptake by both types of cells (60-70%) with no significant differences. Intracellular presence of siRNA was short (∼24 hours) irrespective of the carrier used. Using P-gp specific siRNA, Lipofectamine TM 2000 led to the highest down-regulation of P-gp (∼80%), while polymeric carriers yielded ∼40-55% P-gp downregulation. The polymers, however, were able to protect the siRNA against serum to a better extent than LipofecatmaineTM. Conclusions: The amount of siRNA uptake into cells non-viral carriers is considerably high, which led to significant down-regulation of P-gp. Although siRNA has a short half-life inside the cells, this was sufficient for P-gp inhibition. Future studies are planned to probe correlations between drug accumulation and P-gp inhibition, as well as identifying the most effective carrier for such a siRNA delivery.
290. Improved Gene Transfer into Renal Carcinoma Cells Using an Adenovirus Vector
Shuji Terao,1 Bishnu Acharya,1 Toru Suzuki,1 Katsuyuki Hamada,2 Hiroyuki Mizuguchi,3 Akinobu Gotoh.1 1 Laboratory of Cell and Gene Therapy, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; 2Department of Obstetrics and Gynecology, Ehime University School of Medicine, Toon, Japan; 3Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. Purpose: The transduction efficacy of adenovirus serotype 5 (Ad5) vector in human renal carcinoma cells is generally extremely low due to the non-expression of Coxsackie and adenoviral receptor (CAR), for which Ad5 has a high affinity. We investigated whether the histone deacetylase inhibitor, valproic acid (VPA), or fiber-modified Ad vector containing an RGD motif in the HI loop of the Ad fiber knob could increase the transduction efficiency of Ad5 in human renal carcinoma cells in vitro. Methods: We have tested human renal carcinoma cell lines Caki-1, ACHN, RCC4-VHL, and 786-O, and human bladder S113