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329. A Genetically Engineered Multifunctional Vector for HER2 Targeted Cancer Suicide Gene Therapy
CANCER-TARGETED GENE & CELL THERAPY placed just above the knee. Infusion of normal saline was carried out with a Belmont FMS 2000 Rapid Infuser. Cardiac function was continously monitored by echocardiography while observing for any intracardiac microcavitations indicative of saline leakage during the infusion. In subject 1, analgesia and anxiolysis was provided with fentanyl and midazolam. In subjects 2-4, a combination of fentanyl, midazolam and propofol was administered. No subject complained of any post procedure pain other than due to needle punctures. No adverse safety events occurred in any of the monitored parameters. Conclusion: We have demonstrated that high-pressure retrograde transvenous limb perfusion with saline up to 15% of limb volume at these infusion parameters is safe and feasible. We will escalate the volume to a maximum of 40% of limb volume to determine the maximum safe perfusion parameters and then document the effectiveness of different infusion parameters to produce entry of uid into muscle by T2 MRI. These studies will serve as a basis for future gene therapy clinical trials. Age Diagnosis 31 35 38 27
LGMD2A LGMD2A LGMD Becker MD
Tourniquet Pressure (mm Hg) 310 310 310 310
Infusion Parameters Max Infusion Volume Line Pressure (% limb) (mm Hg) 290 5 300 5 300 10 295 15
Volume (mL)
Flow Rate (mL/min)
245 200 700 975
60 67 70 80
Cancer-Targeted Gene & Cell Therapy 329. A Genetically Engineered Multifunctional Vector for HER2 Targeted Cancer Suicide Gene Therapy Yuhua Wang,1 Arash Hate.1 Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.
1
Background: A recombinant multifunctional vector, namely GHT was bioengineered to target and deliver plasmid DNA (pDNA) to HER2 positive cancer cells. The vector is composed of multiple functional motifs including: 1) a multimerized histone H2A peptide to condense pDNA, 2) a HER2 targeting motif to recognize HER2 positive cancer cells, and 3) a pH-responsive fusogenic peptide to disrupt endosome membranes. Using a reporter gene (GFP), we have previously demonstrated that the vector is able to target and efciently transfect HER2 positive SKOV-3 cells in vitro. In this study, we evaluated the ability of the vector to efciently deliver a cancer suicide gene (pTKSR39) encoding mutant Herpes Simplex Virus Thymidine Kinase to SKOV3 xenograft tumors. Methods: Nanocarrier characterization: Vector GHT was complexed with pTK-SR39 to form nanosize particles. The particle size and surface charge was determined by Malvern Nano ZS90 zetasizer. In vitro cell transfection: GHT/ pTK-SR39 nanocarriers were used to transfect SKOV3 cells in vitro. The expression of TK-SR39 gene in transfected SKOV-3 cells was determined by westernblot analysis using anti-TK-SR39 polyclonal antibody. In vitro cell killing efciency: The acute toxicity of the GHT/ pTK-SR39 nanocarriers was evaluated in SKOV3 cancer cells by a WST-1 cell toxicity assay. The chronic cell toxicity (long term growth inhibition) was examined using a clonogenic assay. In vivo efcacy: GHT/pTK-SR39 nanocarriers (various doses) and controls (total of 9 groups) were injected intratumorally into nude mice bearing SKOV3 xenografts followed by administration of 20mg/kg ganciclovir (GCV). Administration of GCV was varied from 7 to 21 days post transfection and tumor sizes were measured every 5 days. Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Results: GHT was able to condense pTK-SR39 into particles with 59±2nm size and 20±1 mV surface charge. Westernblot analysis demonstrated the expression of pTK-SR39 in SKOV3 cells after transfection. WST1 cell toxicity assay indicated that GHT/pTK-SR39 nanocarriers only in combination with GCV are able to kill SKOV3 cells (up to 87%). The clonogenic assay demonstrated that ca. 89% of SKOV3 were growth inhibited after treatment with GHT/pTK-SR39 plus GCV. The results of animal studies demonstrated that treatment of animals with control groups GCV, PBS, pTK-SR39, and pTK-SR39 plus GCV (protocols 1 to 4) did not inhibit tumor growth. Animals treated with various doses of GHT/pTK-SR39 plus GCV (protocols 5-9) showed signicant tumor size reduction. Animals treated with protocol number 9 where three doses of GHT/pTK-SR39 (equivalent of 5ug) were injected intratumorally on days 1, 3 and 5 followed by administration of GCV for 21 days showed the most promising approach toward tumor size reduction. Conclusion: This study demonstrates the rst successful treatment of xenograft tumors with a recombinant non-viral vector.
330. Comparative Analysis of Enzyme and Pathway Engineering Strategies for Suicide Gene Therapy Applications
Adam J. Johnson,1 Andressa Ardiani,1 Kinta Serve,1 Marilyn Sanchez-Bonilla,2 Margaret E. Black.1,2 1 School of Molecular Biosciences, Washington State University, Pullman, WA; 2Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.
Both bacterial and yeast encoded cytosine deaminases (bCD and yCD, respectively) are highly characterized suicide enzymes used in combination with the prodrug 5-uorocytosine (5FC) for cancer gene therapy applications. However characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. To overcome these limitations, we utilized regio-specic random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD1525) or increased thermostability (yCDdouble, yCDtriple). All mutants exhibited enhanced cancer cell sensitivity to 5FC both in vitro and in vivo as compared to their respective wild-type enzyme. An alternative approach to enhance prodrug activation employs pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, to create bCD/bUPRT or yCD/yUPRT. To further enhance 5FC activation, bCD1525 and yCD mutants were introduced into their respective CD/UPRT constructs and were evaluated in vitro and in vivo for their tumor growth inhibition and bystander killing effects. In these studies, all mutant fusion enzymes enhanced cancer cell sensitivity to 5FC as compared to their mutant CD counterparts, with the bCD1525/bUPRT fusion displaying the greatest reduction in IC50 value of all enzymes or fusions tested. However, in vitro bystander analyses revealed that cells expressing bCD1525/bUPRT lacked signicant bystander cell killing activity whereas bCD1525 exhibited a strong bystander effect. In a mouse xenograft model, tumors expressing bCD1525 displayed superior tumor growth inhibition and bystander activity compared to bCD1525/bUPRT, while tumors expressing yCD mutant or mutant fusion enzyme showed slighter tumor growth restriction and little to no bystander killing. In summary, in contrast to in vitro results that demonstrate the greatest 5FC sensitivity is conferred by bCD1525/ bUPRT, in vivo bCD1525 appears to be the most effective of all enzymes or fusions examined and as such is likely to signicantly improve the clinical outcome of CD/5FC suicide gene therapy applications.
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LGMD2A LGMD2A LGMD Becker MD
Tourniquet Pressure (mm Hg) 310 310 310 310
Infusion Parameters Max Infusion Volume Line Pressure (% limb) (mm Hg) 290 5 300 5 300 10 295 15
Volume (mL)
Flow Rate (mL/min)
245 200 700 975
60 67 70 80
Cancer-Targeted Gene & Cell Therapy 329. A Genetically Engineered Multifunctional Vector for HER2 Targeted Cancer Suicide Gene Therapy Yuhua Wang,1 Arash Hate.1 Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.
1
Background: A recombinant multifunctional vector, namely GHT was bioengineered to target and deliver plasmid DNA (pDNA) to HER2 positive cancer cells. The vector is composed of multiple functional motifs including: 1) a multimerized histone H2A peptide to condense pDNA, 2) a HER2 targeting motif to recognize HER2 positive cancer cells, and 3) a pH-responsive fusogenic peptide to disrupt endosome membranes. Using a reporter gene (GFP), we have previously demonstrated that the vector is able to target and efciently transfect HER2 positive SKOV-3 cells in vitro. In this study, we evaluated the ability of the vector to efciently deliver a cancer suicide gene (pTKSR39) encoding mutant Herpes Simplex Virus Thymidine Kinase to SKOV3 xenograft tumors. Methods: Nanocarrier characterization: Vector GHT was complexed with pTK-SR39 to form nanosize particles. The particle size and surface charge was determined by Malvern Nano ZS90 zetasizer. In vitro cell transfection: GHT/ pTK-SR39 nanocarriers were used to transfect SKOV3 cells in vitro. The expression of TK-SR39 gene in transfected SKOV-3 cells was determined by westernblot analysis using anti-TK-SR39 polyclonal antibody. In vitro cell killing efciency: The acute toxicity of the GHT/ pTK-SR39 nanocarriers was evaluated in SKOV3 cancer cells by a WST-1 cell toxicity assay. The chronic cell toxicity (long term growth inhibition) was examined using a clonogenic assay. In vivo efcacy: GHT/pTK-SR39 nanocarriers (various doses) and controls (total of 9 groups) were injected intratumorally into nude mice bearing SKOV3 xenografts followed by administration of 20mg/kg ganciclovir (GCV). Administration of GCV was varied from 7 to 21 days post transfection and tumor sizes were measured every 5 days. Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Results: GHT was able to condense pTK-SR39 into particles with 59±2nm size and 20±1 mV surface charge. Westernblot analysis demonstrated the expression of pTK-SR39 in SKOV3 cells after transfection. WST1 cell toxicity assay indicated that GHT/pTK-SR39 nanocarriers only in combination with GCV are able to kill SKOV3 cells (up to 87%). The clonogenic assay demonstrated that ca. 89% of SKOV3 were growth inhibited after treatment with GHT/pTK-SR39 plus GCV. The results of animal studies demonstrated that treatment of animals with control groups GCV, PBS, pTK-SR39, and pTK-SR39 plus GCV (protocols 1 to 4) did not inhibit tumor growth. Animals treated with various doses of GHT/pTK-SR39 plus GCV (protocols 5-9) showed signicant tumor size reduction. Animals treated with protocol number 9 where three doses of GHT/pTK-SR39 (equivalent of 5ug) were injected intratumorally on days 1, 3 and 5 followed by administration of GCV for 21 days showed the most promising approach toward tumor size reduction. Conclusion: This study demonstrates the rst successful treatment of xenograft tumors with a recombinant non-viral vector.
330. Comparative Analysis of Enzyme and Pathway Engineering Strategies for Suicide Gene Therapy Applications
Adam J. Johnson,1 Andressa Ardiani,1 Kinta Serve,1 Marilyn Sanchez-Bonilla,2 Margaret E. Black.1,2 1 School of Molecular Biosciences, Washington State University, Pullman, WA; 2Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.
Both bacterial and yeast encoded cytosine deaminases (bCD and yCD, respectively) are highly characterized suicide enzymes used in combination with the prodrug 5-uorocytosine (5FC) for cancer gene therapy applications. However characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. To overcome these limitations, we utilized regio-specic random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD1525) or increased thermostability (yCDdouble, yCDtriple). All mutants exhibited enhanced cancer cell sensitivity to 5FC both in vitro and in vivo as compared to their respective wild-type enzyme. An alternative approach to enhance prodrug activation employs pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, to create bCD/bUPRT or yCD/yUPRT. To further enhance 5FC activation, bCD1525 and yCD mutants were introduced into their respective CD/UPRT constructs and were evaluated in vitro and in vivo for their tumor growth inhibition and bystander killing effects. In these studies, all mutant fusion enzymes enhanced cancer cell sensitivity to 5FC as compared to their mutant CD counterparts, with the bCD1525/bUPRT fusion displaying the greatest reduction in IC50 value of all enzymes or fusions tested. However, in vitro bystander analyses revealed that cells expressing bCD1525/bUPRT lacked signicant bystander cell killing activity whereas bCD1525 exhibited a strong bystander effect. In a mouse xenograft model, tumors expressing bCD1525 displayed superior tumor growth inhibition and bystander activity compared to bCD1525/bUPRT, while tumors expressing yCD mutant or mutant fusion enzyme showed slighter tumor growth restriction and little to no bystander killing. In summary, in contrast to in vitro results that demonstrate the greatest 5FC sensitivity is conferred by bCD1525/ bUPRT, in vivo bCD1525 appears to be the most effective of all enzymes or fusions examined and as such is likely to signicantly improve the clinical outcome of CD/5FC suicide gene therapy applications.
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