- Email: [email protected]
286. Combination of Suicide Gene Therapy with Anti-Angiogenic Gene Therapy Improves Survival in BT4C Rat Glioma
CANCER - TARGETED GENE THERAPY I supporting the potential of the scFv adapter to enhance EGFR-positive tumor specificity of HSV vectors.
285. Development of a Novel Cationic Biopolymer for Targeted Cancer Gene Therapy
Brenda F. Canine,1 Arash Hatefi.1 Pharmaceutical Sciences, Washington State University, Pullman, WA.
1
Background: The goal of cancer gene therapy is to deliver genetic material to tumor cells leading ultimately to tumor cell death. Targeted delivery of the genetic material is preferred to increase treatment efficacy and reduce toxicity. In this study recombinant engineering techniques were used to create a biopolymer carrier that contains (1) a pH sensitive fusogenic peptide (FP) to aid in endosomal escape, 2) a pDNA condensing motif (DCM) rich in arginine and histidine repeating units, 3) a nuclear localization signal (NLS) to direct the genetic material to the nucleus, and 4) a targeting motif (TM) which is a ligand for the HER2 receptor. The engineered vector namely, FP-DCM-NLS-TP was designed to have proteolytic cleavage sites in its sequence to promote polymer degradation once inside cellular compartments. A cathepsin D substrate was also engineered between the NLS and TP domains to promote dissociation of the TP inside endosomes. Methods: Cloning, Expression, Purification, and Characterization: The FP-DCM-NLS-TP gene was synthesized, cloned into the pET21b vector, transformed into BL21 pLysS cells, and purified by Ni-NTA affinity chromatography. The purity was confirmed by SDS-PAGE, western blot, and MALDI-TOF analysis. Incubation with cathepsin D confirmed proteolytic cleavage sites. The biopolymer was condensed with pDNA and the mean hydrodynamic sizes and surface charge was determined by Malvern ZS90 zetasizer. Gel mobility assays evaluated biopolymer/pDNA interactions via agarose gel electrophoresis. Cell Culture and Transfection: SKOV3 or MDA-MB-231 cells were seeded and transfected with vector/ pEGFP complexes. The green fluorescent protein was visualized using an epifluorescent microscope and the percent transfected cell was determined by a flowcytometer. Results: SDS-PAGE and western blot analysis confirmed expression and purification of the biopolymer. MALDI-TOF analysis confirmed the molecular weight of the purified biopolymer. Proteolytic assays verified cleavage by cathepsin D. Hemolytic assay at various pH values illustrated the functionality of the fusogenic peptide. The biopolymer was complexed with pDNA and the size of the particles was determined to be less than 100 nm, which is a suitable size for uptake by receptor mediated endocytosis. Gel mobility assays indicated that the vector interacted with pDNA and the complexes migration was retarded in a dose dependent manner. In the transfection assay, a biopolymer without the NLS, namely FP-DCM-TP was used as a control to test the effects of the NLS on transfection efficiency. pEGFP was complexed with FPDCM-NLS-TP and FP-DCM-TP and used to transfect SKOV-3 cells. Chloroquine treatment resulted in no change in transfection efficiency while addition in baflomycin and nocodazole resulted in significantly reduced transfection efficiency. To test the biopolymers ability to target HER2, SKOV-3 (HER2 positive) and MDA- MB-231(HER2 negative) were transfected with biopolymer/pEGFP complexes. MB-231 had significantly lower GFP expression in comparison to SKOV-3 cells. Conclusion: In conclusion, it was demonstrated that the FP-DCM-NLS-TP biopolymer was able to condense pDNA into nanoparticles, promote endosome disruption, and target cancer cells overexpressing HER2.
S112
286. Combination of Suicide Gene Therapy with Anti-Angiogenic Gene Therapy Improves Survival in BT4C Rat Glioma
Haritha Samaranayake,1,2 Jere Pikkarainen,1,2 Thuomas Mäntylä,1 Thomas Wirth,1 Seppo Ylä-Herttuala.2,3 1 Ark Therapeutics Oy, Kuopio, Finland; 2Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, Kuopio, Finland; 3Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland. Malignant gliomas constitute the majority of primary brain tumors. Conventional therapeutic options provide little hope for these patients. The most aggressive form; glioblastoma multiforme (GBM) has a mean survival of 5-8 months after diagnosis. Gene therapy has evolved as a new therapeutic approach to address this problem with many different strategies being studied at both preclinical as well as clinical trial level. Adenovirus encoding Herpes simplex virus type-1, thymidine kinase (AdHSV-tk) gene therapy with pro-drug ganciclovir (GCV) administration has been the most successful of such approaches with an increase in the mean survival in operable glioma patients, almost up to 15 months in clinical trials. High vascularity of malignant gliomas with increased expression of vascular endothelial growth factor makes these tumours ideal target for anti-angiogenic therapy. Soluble vascular endothelial growth factor receptor-1 has been used successfully to inhibit tumour angiogenesis in several preclinical studies. We hypothesised that the efficacy of AdHSV-tk/GCV suicide gene therapy could be enhanced by combining it with adenovirus encoding soluble vascular endothelial growth factor receptor-1 (AdsVEGFR1) anti-angiogenic gene therapy. The hypothesis was tested in immunocompetent, syngenic BDIX rats bearing intracranial BT4C rat gliomas. AdsVEGFR1 before AdHSV-tk/GCV, simultaneous combination of the two gene therapies and anti-angiogenic gene therapy after suicide gene therapy, were compared against the untreated controls and AdHSV-tk/GCV only group. Kaplan-Meier survival analysis revealed a statistically significant improvement in survival compared to the untreated controls when both gene therapies were administered simultaneously (p-value 0.0263), and when suicide gene therapy was given a week before anti-angiogenic gene therapy (p-value 0.0399). However, in spite of having better median survival in the simultaneous combination and AdHSV-tk/GCV before AdsVEGFR1 groups, compared to the AdHSV-tk/GCV group; these differences did not reach statistical significance. This study revealed that this gene therapy combination is safe and significantly improved the survival. It also demonstrated that adenovirus vectors can be administered repeatedly to immunocompetant rats, without major adverse effects. Further large scale studies and in-depth analyses are needed to ascertain the synergistic effects of this gene therapy combination.
287. Phase I Clinical Trial and U.S. Regulatory Experience with Designer T Cells in Cancer Richard P. Junghans. Division of Surgical Research, Roger Williams Medical Center, Providence, RI.
R P Junghans, Departments of Surgery and Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, RI 02908. The process of bringing any product from the bench to the bedside is complex, and particularly so for gene therapy applications. Yet these are some of the most promising agents for the future treatment of cancer and infectious diseases. Designer T cells are autologous T cells retrovirally-modified to express chimeric immune receptors (CIR) against tumor or viral antigens. In this is described our Phase I clinical trial experience with anti-carcinoembryonic antigen (CEA) designer T cells in adenocarcinoma (1st generation, completed; 2nd generation, enrolling patients) and the regulatory Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
285. Development of a Novel Cationic Biopolymer for Targeted Cancer Gene Therapy
Brenda F. Canine,1 Arash Hatefi.1 Pharmaceutical Sciences, Washington State University, Pullman, WA.
1
Background: The goal of cancer gene therapy is to deliver genetic material to tumor cells leading ultimately to tumor cell death. Targeted delivery of the genetic material is preferred to increase treatment efficacy and reduce toxicity. In this study recombinant engineering techniques were used to create a biopolymer carrier that contains (1) a pH sensitive fusogenic peptide (FP) to aid in endosomal escape, 2) a pDNA condensing motif (DCM) rich in arginine and histidine repeating units, 3) a nuclear localization signal (NLS) to direct the genetic material to the nucleus, and 4) a targeting motif (TM) which is a ligand for the HER2 receptor. The engineered vector namely, FP-DCM-NLS-TP was designed to have proteolytic cleavage sites in its sequence to promote polymer degradation once inside cellular compartments. A cathepsin D substrate was also engineered between the NLS and TP domains to promote dissociation of the TP inside endosomes. Methods: Cloning, Expression, Purification, and Characterization: The FP-DCM-NLS-TP gene was synthesized, cloned into the pET21b vector, transformed into BL21 pLysS cells, and purified by Ni-NTA affinity chromatography. The purity was confirmed by SDS-PAGE, western blot, and MALDI-TOF analysis. Incubation with cathepsin D confirmed proteolytic cleavage sites. The biopolymer was condensed with pDNA and the mean hydrodynamic sizes and surface charge was determined by Malvern ZS90 zetasizer. Gel mobility assays evaluated biopolymer/pDNA interactions via agarose gel electrophoresis. Cell Culture and Transfection: SKOV3 or MDA-MB-231 cells were seeded and transfected with vector/ pEGFP complexes. The green fluorescent protein was visualized using an epifluorescent microscope and the percent transfected cell was determined by a flowcytometer. Results: SDS-PAGE and western blot analysis confirmed expression and purification of the biopolymer. MALDI-TOF analysis confirmed the molecular weight of the purified biopolymer. Proteolytic assays verified cleavage by cathepsin D. Hemolytic assay at various pH values illustrated the functionality of the fusogenic peptide. The biopolymer was complexed with pDNA and the size of the particles was determined to be less than 100 nm, which is a suitable size for uptake by receptor mediated endocytosis. Gel mobility assays indicated that the vector interacted with pDNA and the complexes migration was retarded in a dose dependent manner. In the transfection assay, a biopolymer without the NLS, namely FP-DCM-TP was used as a control to test the effects of the NLS on transfection efficiency. pEGFP was complexed with FPDCM-NLS-TP and FP-DCM-TP and used to transfect SKOV-3 cells. Chloroquine treatment resulted in no change in transfection efficiency while addition in baflomycin and nocodazole resulted in significantly reduced transfection efficiency. To test the biopolymers ability to target HER2, SKOV-3 (HER2 positive) and MDA- MB-231(HER2 negative) were transfected with biopolymer/pEGFP complexes. MB-231 had significantly lower GFP expression in comparison to SKOV-3 cells. Conclusion: In conclusion, it was demonstrated that the FP-DCM-NLS-TP biopolymer was able to condense pDNA into nanoparticles, promote endosome disruption, and target cancer cells overexpressing HER2.
S112
286. Combination of Suicide Gene Therapy with Anti-Angiogenic Gene Therapy Improves Survival in BT4C Rat Glioma
Haritha Samaranayake,1,2 Jere Pikkarainen,1,2 Thuomas Mäntylä,1 Thomas Wirth,1 Seppo Ylä-Herttuala.2,3 1 Ark Therapeutics Oy, Kuopio, Finland; 2Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, Kuopio, Finland; 3Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland. Malignant gliomas constitute the majority of primary brain tumors. Conventional therapeutic options provide little hope for these patients. The most aggressive form; glioblastoma multiforme (GBM) has a mean survival of 5-8 months after diagnosis. Gene therapy has evolved as a new therapeutic approach to address this problem with many different strategies being studied at both preclinical as well as clinical trial level. Adenovirus encoding Herpes simplex virus type-1, thymidine kinase (AdHSV-tk) gene therapy with pro-drug ganciclovir (GCV) administration has been the most successful of such approaches with an increase in the mean survival in operable glioma patients, almost up to 15 months in clinical trials. High vascularity of malignant gliomas with increased expression of vascular endothelial growth factor makes these tumours ideal target for anti-angiogenic therapy. Soluble vascular endothelial growth factor receptor-1 has been used successfully to inhibit tumour angiogenesis in several preclinical studies. We hypothesised that the efficacy of AdHSV-tk/GCV suicide gene therapy could be enhanced by combining it with adenovirus encoding soluble vascular endothelial growth factor receptor-1 (AdsVEGFR1) anti-angiogenic gene therapy. The hypothesis was tested in immunocompetent, syngenic BDIX rats bearing intracranial BT4C rat gliomas. AdsVEGFR1 before AdHSV-tk/GCV, simultaneous combination of the two gene therapies and anti-angiogenic gene therapy after suicide gene therapy, were compared against the untreated controls and AdHSV-tk/GCV only group. Kaplan-Meier survival analysis revealed a statistically significant improvement in survival compared to the untreated controls when both gene therapies were administered simultaneously (p-value 0.0263), and when suicide gene therapy was given a week before anti-angiogenic gene therapy (p-value 0.0399). However, in spite of having better median survival in the simultaneous combination and AdHSV-tk/GCV before AdsVEGFR1 groups, compared to the AdHSV-tk/GCV group; these differences did not reach statistical significance. This study revealed that this gene therapy combination is safe and significantly improved the survival. It also demonstrated that adenovirus vectors can be administered repeatedly to immunocompetant rats, without major adverse effects. Further large scale studies and in-depth analyses are needed to ascertain the synergistic effects of this gene therapy combination.
287. Phase I Clinical Trial and U.S. Regulatory Experience with Designer T Cells in Cancer Richard P. Junghans. Division of Surgical Research, Roger Williams Medical Center, Providence, RI.
R P Junghans, Departments of Surgery and Medicine, Boston University School of Medicine, Roger Williams Medical Center, Providence, RI 02908. The process of bringing any product from the bench to the bedside is complex, and particularly so for gene therapy applications. Yet these are some of the most promising agents for the future treatment of cancer and infectious diseases. Designer T cells are autologous T cells retrovirally-modified to express chimeric immune receptors (CIR) against tumor or viral antigens. In this is described our Phase I clinical trial experience with anti-carcinoembryonic antigen (CEA) designer T cells in adenocarcinoma (1st generation, completed; 2nd generation, enrolling patients) and the regulatory Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy