- Email: [email protected]
71. Intratumoral Delivery of Small Interfering RNA for Inhibition of Tumor Progression in Mice
CANCER-TARGETED GENE THERAPY: NON ADENOVIRAL TARGETING OF VECTORS AND NON VIRAL VECTORS targeted siEFBP markedly inhibits EFT tumor growth when administered early in the course of disease, with little or no tumor growth in many animals. Treatments with vehicle alone, siEFBP alone, formulated unrelated siRNA or formulated siEFBP without the transferrin targeting ligand had no therapeutic effect. The lack of changes in complete blood chemistries, no increases in interferonalpha and IL-12 and major organ pathology show that the treatments do not elicit an immune response and are safe. To our knowledge the first study to show that systemic, non-viral administration of siRNA can inhibit disseminated tumor growth in a sequence-specific manner. M.E.D. is a consultant to and has a financial interest in Insert Therapeutics, Inc.
In conclusion, our data suggest one solution to the question how the in vivo intratumoral delivery of siRNA can be achieved, and that silencing β-catenin or HIF1α by RNAi can be of use in the treatment of cancer.
72. Capsid Modifications Overcome Low Heterogeneous Expression of Heparan Sulfate Proteoglycan That Limits AAV2-Mediated Gene Transfer and Therapeutic Efficacy in Human Ovarian Carcinoma Jeffrey S. Bartlett,1,2 Wenfang Shi.2 Gene Therapy Center, Children’s Research Institute, Columbus, OH; 2Department of Pediatrics, The Ohio State University, Columbus, OH.
1
71. Intratumoral Delivery of Small Interfering RNA for Inhibition of Tumor Progression in Mice Yuki Takahashi,1 Makiya Nishikawa,1 Yoshinobu Takakura.1 Department of Biopharmaceutics and Drug Metabolism, Grad. Sch. of Pharm. Sci., Kyoto University, Kyoto, Japan.
1
RNA interference (RNAi) is a post-transcriptional gene silencing event in which short double-stranded RNA (siRNA) degrades target mRNA in a sequence-specific manner. Silencing oncogenes or other genes contributing to tumor cell malignancy or progression by siRNA or siRNA-expressing vector offers a therapeutic treatment for cancer patients. For treating cancer patients with RNAi effector, its delivery to tumor cells is one of the key factors, because the gene silencing event is limited in the cells reached by the RNAi effector. To examine whether siRNA or siRNA-expressing vector is effective in reducing target gene expression in tumor cells in vivo, we developed tumor cell lines that stably express reporter genes for a convenient, sensitive and quantitative evaluation of the RNAi effect. Namely, mouse melanoma B16-BL6 cells were stably transfected with firefly (model target gene of RNAi) and sea pansy (indicator of tumor cell number) luciferases to obtain B16-BL6/dual Luc cells. We found that the ratio of the luciferase activities in the tumor cells can be used as an indicator of RNAi. When the siRNA-expressing plasmid DNA (pDNA) was added to the cells, the luciferase activity decreased with time and reached a trough level at 2 days. Then, the activity returned to the initial level at 12 days. siRNA also showed a similar inhibitory profile, but the decrease and recovery in the luciferase activity were faster than those of siRNA-expressing pDNA. Then, the B16-BL6/dual Luc cells were inoculated into the footpad of mice to examine whether the luciferase expression is suppressed by the delivery of RNAi effectors in vivo. A single injection of either siRNA or siRNA-expressing pDNA into the tumor tissue followed by electroporation (1000 V/cm, 5 ms, 4 Hz, 12 pulses) reduced the luciferase activity to about 30% of the control value. Next, we investigated whether silencing the expression of genes related to tumor cell progression by RNAi can inhibit the tumor cell growth. To this end, β-catenin and hypoxia-inducible factor 1α(HIF1α) were selected as the target. Transfection of siRNA-expressing pDNA targeting to b-catenin or HIF1a reduced each target gene expression in cultured B16 cells to about 20% (β-catenin) and 25% (HIF1α) at mRNA level compared with those in B16 cells transfected with pDNA expressing no effective siRNA. In addition, the treatment resulted in the decrease in the number of B16 cells to about 50% (βcatenin) and 70% (HIF1α) of a control value at 3 days. Then we investigated whether the intratumoral delivery of siRNA-expressing pDNA targeting to β-catenin or HIF1α can be a therapeutic treatment against the proliferation of the tumor cells in mice. To investigate this possibility, we administered the siRNA-expressing pDNA targeting to β-catenin or HIF1α to the tumor-bearing mice by the same procedure as above. As a result, we found that the intratumoral delivery of siRNA-expressing pDNA targeting β-catenin or HIF1α inhibited the tumor growth. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
Ovarian cancer is one of the more common malignancies affecting women in the United States. Our goal has been to develop a genebased therapy for ovarian cancer using AAV as a vector. However, the utility of AAV vectors for ovarian cancer gene therapy is limited by the distribution of heparan sulfate proteoglycan (HSPG), the virus’s primary attachment receptor, on tumor cell populations. In order to achieve HSPG-independent gene delivery, we have shown that the tropism of AAV can be expanded by genetically altering the viral capsid. We have analyzed tumor tissue from 40 patients with peritoneal ovarian carcinoma for expression of HSPG and other receptors. We found that only about 65% of human tumors express the appropriate HSPG epitope required for AAV infection, whereas greater than 90% express either αvβ3 or αvβ5 integrins. We also examined a panel of 6 different ovarian cancer cell lines (PA-1, OV4, OVCAR3, Hey, OV3 and SKOV-3) and similarly found that only half of these cell lines expressed HSPG, yet all expressed either αvβ3 or αvβ5 integrins. Not surprisingly, those cell lines that failed to express HSPG were not transduced with AAV2-based vectors. We have shown that incorporation of an Arg-Gly-Asp (RGD) – containing peptide at sites within the AAV capsid enables vectors to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying this peptide ligand were produced to wild-type titer and shown to specifically target integrin receptors on ovarian cancer cells. Furthermore, we show that these modified vectors actually mediate infection via this engineered interaction. We report significant increases in gene transfer to previously non-permissive ovarian cancer cell lines, suggesting that AAV vectors displaying RGD peptides may be of utility for treatment of neoplasms characterized by a deficiency of HSPG expression. We next tested these vectors for efficient transduction of ovarian carcinoma in vivo. Tumors were established by intraperitoneal injection of SKOV-3 cells in SCID mice. Either RGD-AAV2 vector or unmodified AAV2 vector expressing GFP or LacZ marker genes was administered to the peritoneal cavity five days after tumors were established. Animals were sacrificed at various times post treatment and gene delivery and expression were evaluated by immunostaining and enzymatic activity. Both assays showed that marker gene transfer and expression was nearly two-orders of magnitude greater in RGDAAV treated animals than in animals treated with unmodified AAV vector. Importantly, the modified AAV vectors did not transduce normal mesothelial tissue or other internal organs. RGD-modified AAV2 vectors encoding HSV-TK have been developed and tested in vitro and in vivo. Incorporation of the RGD tumor-targeting motif in these vectors significantly increased anti-tumor toxicity in vitro and efficacy in vivo. These results suggest that RGD-modified AAV vectors may offer significant therapeutic advantages for ovarian cancer gene therapy.
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In conclusion, our data suggest one solution to the question how the in vivo intratumoral delivery of siRNA can be achieved, and that silencing β-catenin or HIF1α by RNAi can be of use in the treatment of cancer.
72. Capsid Modifications Overcome Low Heterogeneous Expression of Heparan Sulfate Proteoglycan That Limits AAV2-Mediated Gene Transfer and Therapeutic Efficacy in Human Ovarian Carcinoma Jeffrey S. Bartlett,1,2 Wenfang Shi.2 Gene Therapy Center, Children’s Research Institute, Columbus, OH; 2Department of Pediatrics, The Ohio State University, Columbus, OH.
1
71. Intratumoral Delivery of Small Interfering RNA for Inhibition of Tumor Progression in Mice Yuki Takahashi,1 Makiya Nishikawa,1 Yoshinobu Takakura.1 Department of Biopharmaceutics and Drug Metabolism, Grad. Sch. of Pharm. Sci., Kyoto University, Kyoto, Japan.
1
RNA interference (RNAi) is a post-transcriptional gene silencing event in which short double-stranded RNA (siRNA) degrades target mRNA in a sequence-specific manner. Silencing oncogenes or other genes contributing to tumor cell malignancy or progression by siRNA or siRNA-expressing vector offers a therapeutic treatment for cancer patients. For treating cancer patients with RNAi effector, its delivery to tumor cells is one of the key factors, because the gene silencing event is limited in the cells reached by the RNAi effector. To examine whether siRNA or siRNA-expressing vector is effective in reducing target gene expression in tumor cells in vivo, we developed tumor cell lines that stably express reporter genes for a convenient, sensitive and quantitative evaluation of the RNAi effect. Namely, mouse melanoma B16-BL6 cells were stably transfected with firefly (model target gene of RNAi) and sea pansy (indicator of tumor cell number) luciferases to obtain B16-BL6/dual Luc cells. We found that the ratio of the luciferase activities in the tumor cells can be used as an indicator of RNAi. When the siRNA-expressing plasmid DNA (pDNA) was added to the cells, the luciferase activity decreased with time and reached a trough level at 2 days. Then, the activity returned to the initial level at 12 days. siRNA also showed a similar inhibitory profile, but the decrease and recovery in the luciferase activity were faster than those of siRNA-expressing pDNA. Then, the B16-BL6/dual Luc cells were inoculated into the footpad of mice to examine whether the luciferase expression is suppressed by the delivery of RNAi effectors in vivo. A single injection of either siRNA or siRNA-expressing pDNA into the tumor tissue followed by electroporation (1000 V/cm, 5 ms, 4 Hz, 12 pulses) reduced the luciferase activity to about 30% of the control value. Next, we investigated whether silencing the expression of genes related to tumor cell progression by RNAi can inhibit the tumor cell growth. To this end, β-catenin and hypoxia-inducible factor 1α(HIF1α) were selected as the target. Transfection of siRNA-expressing pDNA targeting to b-catenin or HIF1a reduced each target gene expression in cultured B16 cells to about 20% (β-catenin) and 25% (HIF1α) at mRNA level compared with those in B16 cells transfected with pDNA expressing no effective siRNA. In addition, the treatment resulted in the decrease in the number of B16 cells to about 50% (βcatenin) and 70% (HIF1α) of a control value at 3 days. Then we investigated whether the intratumoral delivery of siRNA-expressing pDNA targeting to β-catenin or HIF1α can be a therapeutic treatment against the proliferation of the tumor cells in mice. To investigate this possibility, we administered the siRNA-expressing pDNA targeting to β-catenin or HIF1α to the tumor-bearing mice by the same procedure as above. As a result, we found that the intratumoral delivery of siRNA-expressing pDNA targeting β-catenin or HIF1α inhibited the tumor growth. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
Ovarian cancer is one of the more common malignancies affecting women in the United States. Our goal has been to develop a genebased therapy for ovarian cancer using AAV as a vector. However, the utility of AAV vectors for ovarian cancer gene therapy is limited by the distribution of heparan sulfate proteoglycan (HSPG), the virus’s primary attachment receptor, on tumor cell populations. In order to achieve HSPG-independent gene delivery, we have shown that the tropism of AAV can be expanded by genetically altering the viral capsid. We have analyzed tumor tissue from 40 patients with peritoneal ovarian carcinoma for expression of HSPG and other receptors. We found that only about 65% of human tumors express the appropriate HSPG epitope required for AAV infection, whereas greater than 90% express either αvβ3 or αvβ5 integrins. We also examined a panel of 6 different ovarian cancer cell lines (PA-1, OV4, OVCAR3, Hey, OV3 and SKOV-3) and similarly found that only half of these cell lines expressed HSPG, yet all expressed either αvβ3 or αvβ5 integrins. Not surprisingly, those cell lines that failed to express HSPG were not transduced with AAV2-based vectors. We have shown that incorporation of an Arg-Gly-Asp (RGD) – containing peptide at sites within the AAV capsid enables vectors to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying this peptide ligand were produced to wild-type titer and shown to specifically target integrin receptors on ovarian cancer cells. Furthermore, we show that these modified vectors actually mediate infection via this engineered interaction. We report significant increases in gene transfer to previously non-permissive ovarian cancer cell lines, suggesting that AAV vectors displaying RGD peptides may be of utility for treatment of neoplasms characterized by a deficiency of HSPG expression. We next tested these vectors for efficient transduction of ovarian carcinoma in vivo. Tumors were established by intraperitoneal injection of SKOV-3 cells in SCID mice. Either RGD-AAV2 vector or unmodified AAV2 vector expressing GFP or LacZ marker genes was administered to the peritoneal cavity five days after tumors were established. Animals were sacrificed at various times post treatment and gene delivery and expression were evaluated by immunostaining and enzymatic activity. Both assays showed that marker gene transfer and expression was nearly two-orders of magnitude greater in RGDAAV treated animals than in animals treated with unmodified AAV vector. Importantly, the modified AAV vectors did not transduce normal mesothelial tissue or other internal organs. RGD-modified AAV2 vectors encoding HSV-TK have been developed and tested in vitro and in vivo. Incorporation of the RGD tumor-targeting motif in these vectors significantly increased anti-tumor toxicity in vitro and efficacy in vivo. These results suggest that RGD-modified AAV vectors may offer significant therapeutic advantages for ovarian cancer gene therapy.
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