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II Trial Evaluating Combined Radiotherapy and In-Situ Gene-Therapy; Interim Results
ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER DNA laddering were used to explore the possible mechanism of cell death induced by cell fusion or viral proteins. Positive signs of apoptosis were observed both in fusing cells infected by Sinrep/ GALV.fus and in nonfusing cells infected by Sinrepβgal. To investigate the production of the infectious microparticles, we transfected U87 cells with Sinrep5GALV.fus and Sinrep5LacZ alone or the two plasmids together without helper plasmid. Supernatants and cell homogenates were prepared and filtered through 0.45mm membrane, and used to infect fresh U87 cells. Infectious particles were released from all plasmid-transfected cells, as evidenced by low level transfer of functional GALV.fus or β-galactosidase genes. Treatment of these infectious microparticles with RNase had no effect on their infectivity. Sinrep5GALV.fus viruses were subsequently used to treat intracerebral U87 glioma xenografts in nude mice and led to significantly improved survival rates in the vector-treated animals.
722. Phase I/II Trial Evaluating Combined Radiotherapy and In-Situ Gene-Therapy; Interim Results Bin S. Teh,1 Laura K. Aguilar,4 Maria T. Vlachaki,1 Gustavo E. Ayala,2 Thomas M. Wheeler,2 Wei Y. Mai,2 James Caillouet,1 Maria Davis,1 Larry S. Carpenter,1 Hsin H. Lu,1 Joseph K. Chiu,1 Shiao Y. Woo,1 Brian J. Miles,3 Dov Kadmon,3 Timothy C. Thompson,3 Edward B. Butler,1 Estuardo Aguilar-Cordova.5 1 Radiation Oncology, Baylor College of Medicine, Houston, TX, United States; 2Pathology, Baylor College of Medicine, Houston, TX, United States; 3Scott Department of Urology, Baylor College of Medicine, Houston, TX, United States; 4Harvard Gene Therapy Initiative, Harvard Medical School, Boston, MA, United States; 5 Advantagene, Inc., Boston, MA, United States. Patients with newly diagnosed prostate cancer are being evaluated in this clinical trial in which gene therapy is added to standard-ofcare radiation and hormonal therapy. This study is based on animal tumor models demonstrating improved outcomes from gene therapy combined with radiation. Gene therapy consists of transrectal ultrasound guided intratumoral injection of 5x1011 particles of an adenoviral vector expressing herpes simplex virus thymidine kinase (AdV-tk) followed by oral administration of the prodrug valacyclovir for 14 days. Intensity modulated radiation therapy (IMRT) is used for all patients. The study is divided into three arms based on clinical disease categorization and the associated appropriate standard-ofcare therapy. Over 60 patients have been treated to date with at least 20 patients being followed for 2 years after treatment. Median age was 68 years (39 -85). Median follow-up for the entire group was 16.6 months (1.4 -33.8 months). No significant toxicity has been observed to date and compliance has been excellent. Patients in Arm A (low risk), who begin radiation therapy within 48-72 hours of receiving the first gene therapy injection, had a transient rise in PSA at two weeks which was significantly greater than that observed in a control group treated with radiation alone. At 27 months follow up, 8 of 8 patients in Arm A had a PSA ≤ 1 as compared to 2 of 4 control patients. Patients in Arm B and C begin hormonal ablation at the time of the first gene therapy injection and thus have a rapid fall in PSA without the transient rise as seen in Arm A. In Arm A and B, over 80% of patients have negative biopsy by 5-7 months (time from first AdV-tk injection) and 100% by 21 months. All patients in Arm A (median follow-up16.4 months) and Arm B (median follow-up of 16.9 months) had biochemical control thus far. Arm C patients have positive pelvic lymph nodes. at presentation; 3 of 4 of these patients have developed distant metastases but local disease was controlled. The combination of AdV-tk/valacyclovir gene therapy and radiation has been well
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
tolerated, feasible to deliver as an outpatient procedure to a large number of patients and early results of surrogate markers are encouraging.
723. Bifunctional Chimeric SuperCD Suicide Gene In Vivo Expression Found To Be Highly Effective in a Rat Hepatoma Model Florian Graepler,1 Wolfgang A. Wybranietz,1 Ulrike Schmidt,1 Irina Smirnow,1 Christine D. Gross,1 Martin Spiegel,1 Andrea Schenk,1 Horst Feichtiger,1 Hansjoerg Graf,2 Ulrike A. Lauer,2 Michael Gregor,1 Sorin Armeanu,1 Michael Bitzer,1 Ulrich M. Lauer.1 1 Department of Internal Medicine I, University Clinic Tuebingen, Tuebingen, Germany; 2Section of Experimental Radiology, University Clinic Tuebingen, Tuebingen, Germany. Background & Aims: Low transduction efficiencies of primary and secondary liver tumors are a major limitation in liver tumor suicide gene therapy. For compensation, catalytically superior genedirected enzyme prodrug therapy systems are demanded. Methods: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC) we have directly fused the yeast cytosine deaminase (YCD) suicide gene to a 5´-terminally deleted yeast uracil phospho-ribosyltransferase (YUPRT) gene thereby generating a chimeric bifunctional SuperCD (YCD::YUPRT) suicide gene. Results: In vitro, stably transduced Morris rat hepatoma MH3924A cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) demonstrated a marked enhancement in cell killing under incubation with 5-FC compared with MH cells stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin (MH BCD), respectively. In vivo, MH SuperCD tumors sub-cutaneously implanted into syngeneic ACI rats demonstrated a complete regression under systemic 5-FC application, whereas MH tumors without transgene expression (MH naïve) showed rapid progression. Molecular and functional analysis of explanted MH SuperCD control tumors excluded any relevant selective pressure against the presence of the SuperCD transgene accompanying in vivo passage of SuperCD tumors. Furthermore, MH SuperCD tumors implanted ortho-topically into the liver demonstrated a significant tumor regression under a 10 d systemic 5-FC application (p < 0.01), as measured by non-invasive magnetic resonance imaging. Additionally, a major reduction in 5-FC dosing (twice weekly 5-FC i.p. injections at a dosage of 283 mg/kg) was found to be sufficient to achieve complete regressions of orthotopically implanted MH SuperCD tumors. Conclusions: Bifunctional SuperCD suicide gene expression was found to be highly effective in a rat hepatoma model under both subcutaneous and orthotopic implantation conditions. Concerning current knowledge on severe gene therapeutic side effects, reductions in vector dosages applied to liver tumors potentially could be achieved employing the SuperCD suicide gene thereby constituting an important safety parameter.
724. Retroviral Vectors Pseudotyped with the RD114 Envelope Kill Cancer Cells by Syncytia Formation Emmanuelle F. Germain,1 Gaelle V. Roullin,1 Jian Qiao,1 Manuel P. Caruso.1 1 Gene Therapy Laboratory, Cancer Research Center, 9 McMahon St, Laval University, Quebec, QC, Canada. Background: The knowledge in basic retrovirology has enabled the development of retroviral vectors for gene transfer. These vectors are mainly derived from the MLV and can be engineered to display envelope (env) of other viruses at their surface. This process, called S279
722. Phase I/II Trial Evaluating Combined Radiotherapy and In-Situ Gene-Therapy; Interim Results Bin S. Teh,1 Laura K. Aguilar,4 Maria T. Vlachaki,1 Gustavo E. Ayala,2 Thomas M. Wheeler,2 Wei Y. Mai,2 James Caillouet,1 Maria Davis,1 Larry S. Carpenter,1 Hsin H. Lu,1 Joseph K. Chiu,1 Shiao Y. Woo,1 Brian J. Miles,3 Dov Kadmon,3 Timothy C. Thompson,3 Edward B. Butler,1 Estuardo Aguilar-Cordova.5 1 Radiation Oncology, Baylor College of Medicine, Houston, TX, United States; 2Pathology, Baylor College of Medicine, Houston, TX, United States; 3Scott Department of Urology, Baylor College of Medicine, Houston, TX, United States; 4Harvard Gene Therapy Initiative, Harvard Medical School, Boston, MA, United States; 5 Advantagene, Inc., Boston, MA, United States. Patients with newly diagnosed prostate cancer are being evaluated in this clinical trial in which gene therapy is added to standard-ofcare radiation and hormonal therapy. This study is based on animal tumor models demonstrating improved outcomes from gene therapy combined with radiation. Gene therapy consists of transrectal ultrasound guided intratumoral injection of 5x1011 particles of an adenoviral vector expressing herpes simplex virus thymidine kinase (AdV-tk) followed by oral administration of the prodrug valacyclovir for 14 days. Intensity modulated radiation therapy (IMRT) is used for all patients. The study is divided into three arms based on clinical disease categorization and the associated appropriate standard-ofcare therapy. Over 60 patients have been treated to date with at least 20 patients being followed for 2 years after treatment. Median age was 68 years (39 -85). Median follow-up for the entire group was 16.6 months (1.4 -33.8 months). No significant toxicity has been observed to date and compliance has been excellent. Patients in Arm A (low risk), who begin radiation therapy within 48-72 hours of receiving the first gene therapy injection, had a transient rise in PSA at two weeks which was significantly greater than that observed in a control group treated with radiation alone. At 27 months follow up, 8 of 8 patients in Arm A had a PSA ≤ 1 as compared to 2 of 4 control patients. Patients in Arm B and C begin hormonal ablation at the time of the first gene therapy injection and thus have a rapid fall in PSA without the transient rise as seen in Arm A. In Arm A and B, over 80% of patients have negative biopsy by 5-7 months (time from first AdV-tk injection) and 100% by 21 months. All patients in Arm A (median follow-up16.4 months) and Arm B (median follow-up of 16.9 months) had biochemical control thus far. Arm C patients have positive pelvic lymph nodes. at presentation; 3 of 4 of these patients have developed distant metastases but local disease was controlled. The combination of AdV-tk/valacyclovir gene therapy and radiation has been well
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
tolerated, feasible to deliver as an outpatient procedure to a large number of patients and early results of surrogate markers are encouraging.
723. Bifunctional Chimeric SuperCD Suicide Gene In Vivo Expression Found To Be Highly Effective in a Rat Hepatoma Model Florian Graepler,1 Wolfgang A. Wybranietz,1 Ulrike Schmidt,1 Irina Smirnow,1 Christine D. Gross,1 Martin Spiegel,1 Andrea Schenk,1 Horst Feichtiger,1 Hansjoerg Graf,2 Ulrike A. Lauer,2 Michael Gregor,1 Sorin Armeanu,1 Michael Bitzer,1 Ulrich M. Lauer.1 1 Department of Internal Medicine I, University Clinic Tuebingen, Tuebingen, Germany; 2Section of Experimental Radiology, University Clinic Tuebingen, Tuebingen, Germany. Background & Aims: Low transduction efficiencies of primary and secondary liver tumors are a major limitation in liver tumor suicide gene therapy. For compensation, catalytically superior genedirected enzyme prodrug therapy systems are demanded. Methods: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC) we have directly fused the yeast cytosine deaminase (YCD) suicide gene to a 5´-terminally deleted yeast uracil phospho-ribosyltransferase (YUPRT) gene thereby generating a chimeric bifunctional SuperCD (YCD::YUPRT) suicide gene. Results: In vitro, stably transduced Morris rat hepatoma MH3924A cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) demonstrated a marked enhancement in cell killing under incubation with 5-FC compared with MH cells stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin (MH BCD), respectively. In vivo, MH SuperCD tumors sub-cutaneously implanted into syngeneic ACI rats demonstrated a complete regression under systemic 5-FC application, whereas MH tumors without transgene expression (MH naïve) showed rapid progression. Molecular and functional analysis of explanted MH SuperCD control tumors excluded any relevant selective pressure against the presence of the SuperCD transgene accompanying in vivo passage of SuperCD tumors. Furthermore, MH SuperCD tumors implanted ortho-topically into the liver demonstrated a significant tumor regression under a 10 d systemic 5-FC application (p < 0.01), as measured by non-invasive magnetic resonance imaging. Additionally, a major reduction in 5-FC dosing (twice weekly 5-FC i.p. injections at a dosage of 283 mg/kg) was found to be sufficient to achieve complete regressions of orthotopically implanted MH SuperCD tumors. Conclusions: Bifunctional SuperCD suicide gene expression was found to be highly effective in a rat hepatoma model under both subcutaneous and orthotopic implantation conditions. Concerning current knowledge on severe gene therapeutic side effects, reductions in vector dosages applied to liver tumors potentially could be achieved employing the SuperCD suicide gene thereby constituting an important safety parameter.
724. Retroviral Vectors Pseudotyped with the RD114 Envelope Kill Cancer Cells by Syncytia Formation Emmanuelle F. Germain,1 Gaelle V. Roullin,1 Jian Qiao,1 Manuel P. Caruso.1 1 Gene Therapy Laboratory, Cancer Research Center, 9 McMahon St, Laval University, Quebec, QC, Canada. Background: The knowledge in basic retrovirology has enabled the development of retroviral vectors for gene transfer. These vectors are mainly derived from the MLV and can be engineered to display envelope (env) of other viruses at their surface. This process, called S279