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169. The Creation of Novel Oncolytic Vaccinia Virus Vectors for Efficient Systemic Delivery of Transgenes to Tumors
“OTHER” DNA VIRUS VECTORS 167. Both Oncolysis and Tumor Immunity Are Involved in an Autitumoral Efficacy by Intratumoral Injection of Recombinant Vaccinia Virus (TK Deleted, hGM-CSF Inserted Wyeth Strain) in a VX2 Rabbit Model Joon-Hyeong Kim,1 Byeong-Ho Park,2 Jong-Young Oh,2 David H. Kirn,3 Steve H. Thorne,4 Jae-Seok Kim,5 Tae-Ho Hwang.1 1 Department of Pharmacology, Dong-A University of Medical College, Busan, Republic of Korea; 2Department of Radiology, Dong-A University of Medical College, Busan, Republic of Korea; 3 CEO, JENNEREX Pharm.Inc., San Franscisco, CA; 4Bio-X Program, Dept of Pediatrics, School of Medicine, Stanford Univ., Safransisco, CA; 5Department of Hemato-Oncolgy, Dong-A University of Medical College, Busan, Republic of Korea. Several approaches to gene therapy for cancer have yielded promising results in rodent models. The translation of these results to the clinical realm has been delayed by the lack of tumor models in large animals. We investigated the efficacy of thymidine kinase (TK) deleted human GM-CSF inserted mutants of Wyeth strain of vaccinia virus (JX-594) using in VX-2 rabbit hepatoma model. VX-2 rabbit carcinoma cells were maintained by serial transplantation in the thigh muscles of New Zealand White rabbits, and VX-2 tumor fragment (1mm3) was injected into left hepatic lobe. The primary tumor formation was detected by U/S on week 2 to week 3 post VX-2 injection. JX-594 (10^9 pfu) was injected by U/S guide immediately after tumor detection (<1.5 cm2 in a diameter) into intratumorally (IT) or intravenously, and then CT was taken every a week to monitor hepatoma growth. The tumor growth after both IT or IV injection significantly was significantly inhibited. Furthermore, the metastatic tumor nodule was not observed after IT or IV JX-594 injection while 100 % was observed in rabbits without vv administration. Serum human GM-CSF was maintained to at least one week both in IT and IV injected groups, and the degree of therapeutic efficacy was related with GM-CSF production. Based on in vitro and in vivo tumor-specific replication capacity of JX-594 replication capacity, both direct viral growth and increased tumor immunity by GM-CSF produced by JX-594 might be responsible for antitumoral efficacy and antimetastatic effect. This preclinical study of hepatoma treatment shows JX-594 acts as an oncolytic vaccinia virus suggesting that JX-594 might be potent and safe viral vector for the clinical application for tumor-directed gene therapy.
168. Antitumoral Efficacy of Multiple Injection of JX-594 (Thymidine Kinase (TK) Deleted, Human GM-CSF Inserted Wyeth Strain) Via Tail Vein in NNitrosomorpholine (NNM) Treated Rats Jae-seok Kim,1 June-Hyung Kim,1 Jong-yeong Oh,2 Byeong-ho Park,2 Jin-Han Yoon,2 Steve H. Thorne,3 David H. Kirn,4 Tae-ho Hwang.1 1 MRCCMT, Dong-A University of Medical College, Busan, Republic of Korea; 2Dong-A University of Medical College, Busan, Republic of Korea; 3Bio-X Program, Dept of Pediatrics, School of Medicine, Stanford Univ, San Franscisco, CA; 4 JENNEREX Pharamceutical Inc., San Franscisco, CA.
heterogeneously widespread increased echogenecity), and nodularity (defined as a >0.6-cm-sized echogenic region and clearly showing a tumor-like mass). The gross and histological findings indicated that coarseness and nodularity shown in US reflected fibrosis and hepatocellular carcinoma or cholangiofibroma, respectively. U/S detectable tumor appeared on week 22 post NNM treatment (5>mm in diameter), and rats bearing HCC detected by U/S was injected by JX-594 via tail vein (four times every two weeks, 108 pfu/300 ml for each injection). Based on the similarity of progression of liver disease in NNM treated rat model to that of human liver disease, the clinical benefit of antitumoral efficacy by intravenous injection of JX-594, if it is, could be evaluated. U/S (every two weeks) monitoring showed that significantly decrease of tumor size after tail vein injection of JX-594. Q-PCR results showed indirect evidence of differential viral replication capacity in normal, tumor, and cirrhotic nodules, suggesting oncolytic capacity of JX-594. Eventually, quantification of cirrhosis, ascites and survival benefit could be measured between natural and JX-594 injected ones in NNM treated rats.
169. The Creation of Novel Oncolytic Vaccinia Virus Vectors for Efficient Systemic Delivery of Transgenes to Tumors Steve H. Thorne,1 David H. Kirn.1 1 MMolecular Virology, Jennerex Biotherapeutics. While oncolytic adenoviral vectors, such as ONYX-015, have previously demonstrated anti-tumor potential in both pre-clinical and clinical trials, they have shown limited systemic delivery potential. We have developed a systemic delivery platform based on strains of replication selective vaccinia virus. Vaccinia was chosen as initial comparisons demonstrated that this virus was capable of considerably more efficient and rapid destruction of tumor targets than adenovirus and because even wild type strains displayed an innate tumor targeting potential. Several gene deletions were tested in vitro and found to further increase the tumor selectivity of vaccinia by limiting viral replication to transformed cells. Different deletions have allowed targeting of tumors based on a variety of selection mechanisms. These include the expression of cellular oncogenes, cellular proliferation, immune status of the tumor, tumor vasculature and loss of apoptotic potential within the tumor cell. Several of these deletion strains have since been selected for further development, these include vaccinia vectors targeting proliferating cells and cells with mutations in the Ras pathway and vectors targeting the immune privileged status of the tumor. In vivo studies showed that these viruses could be effectively delivered to tumors systemically, and that systemic delivery was followed by a vigorous anti-tumor response. This was despite the fact that these strains were highly attenuated in non-transformed cells, even in immunodeficient hosts. In addition, molecular imaging was used to demonstrate the ability of these viruses to selectively express transgenes to high levels in tumors following systemic delivery. The large cloning capacity of vaccinia allows for multiple transgenes to be expressed from one viral backbone; genes for increased cytotoxic and bystander effects, genes for imaging and genes to allow shutdown of viral replication as a safety mechanism have therefore been incorporated onto the same vector.
Carcinogen-induced hepatoma in immunocompetent animal models has shown a progress similar to the clinical course of human hepatoma. Ultrasonography (U/S) was used for consecutive evaluation of the phenotypic changes in Sprague-Dawley rats exposed for 8 weeks to NNM (175 mg/L). Three distinctive US findings were ascites, coarseness (defined as small and
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
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168. Antitumoral Efficacy of Multiple Injection of JX-594 (Thymidine Kinase (TK) Deleted, Human GM-CSF Inserted Wyeth Strain) Via Tail Vein in NNitrosomorpholine (NNM) Treated Rats Jae-seok Kim,1 June-Hyung Kim,1 Jong-yeong Oh,2 Byeong-ho Park,2 Jin-Han Yoon,2 Steve H. Thorne,3 David H. Kirn,4 Tae-ho Hwang.1 1 MRCCMT, Dong-A University of Medical College, Busan, Republic of Korea; 2Dong-A University of Medical College, Busan, Republic of Korea; 3Bio-X Program, Dept of Pediatrics, School of Medicine, Stanford Univ, San Franscisco, CA; 4 JENNEREX Pharamceutical Inc., San Franscisco, CA.
heterogeneously widespread increased echogenecity), and nodularity (defined as a >0.6-cm-sized echogenic region and clearly showing a tumor-like mass). The gross and histological findings indicated that coarseness and nodularity shown in US reflected fibrosis and hepatocellular carcinoma or cholangiofibroma, respectively. U/S detectable tumor appeared on week 22 post NNM treatment (5>mm in diameter), and rats bearing HCC detected by U/S was injected by JX-594 via tail vein (four times every two weeks, 108 pfu/300 ml for each injection). Based on the similarity of progression of liver disease in NNM treated rat model to that of human liver disease, the clinical benefit of antitumoral efficacy by intravenous injection of JX-594, if it is, could be evaluated. U/S (every two weeks) monitoring showed that significantly decrease of tumor size after tail vein injection of JX-594. Q-PCR results showed indirect evidence of differential viral replication capacity in normal, tumor, and cirrhotic nodules, suggesting oncolytic capacity of JX-594. Eventually, quantification of cirrhosis, ascites and survival benefit could be measured between natural and JX-594 injected ones in NNM treated rats.
169. The Creation of Novel Oncolytic Vaccinia Virus Vectors for Efficient Systemic Delivery of Transgenes to Tumors Steve H. Thorne,1 David H. Kirn.1 1 MMolecular Virology, Jennerex Biotherapeutics. While oncolytic adenoviral vectors, such as ONYX-015, have previously demonstrated anti-tumor potential in both pre-clinical and clinical trials, they have shown limited systemic delivery potential. We have developed a systemic delivery platform based on strains of replication selective vaccinia virus. Vaccinia was chosen as initial comparisons demonstrated that this virus was capable of considerably more efficient and rapid destruction of tumor targets than adenovirus and because even wild type strains displayed an innate tumor targeting potential. Several gene deletions were tested in vitro and found to further increase the tumor selectivity of vaccinia by limiting viral replication to transformed cells. Different deletions have allowed targeting of tumors based on a variety of selection mechanisms. These include the expression of cellular oncogenes, cellular proliferation, immune status of the tumor, tumor vasculature and loss of apoptotic potential within the tumor cell. Several of these deletion strains have since been selected for further development, these include vaccinia vectors targeting proliferating cells and cells with mutations in the Ras pathway and vectors targeting the immune privileged status of the tumor. In vivo studies showed that these viruses could be effectively delivered to tumors systemically, and that systemic delivery was followed by a vigorous anti-tumor response. This was despite the fact that these strains were highly attenuated in non-transformed cells, even in immunodeficient hosts. In addition, molecular imaging was used to demonstrate the ability of these viruses to selectively express transgenes to high levels in tumors following systemic delivery. The large cloning capacity of vaccinia allows for multiple transgenes to be expressed from one viral backbone; genes for increased cytotoxic and bystander effects, genes for imaging and genes to allow shutdown of viral replication as a safety mechanism have therefore been incorporated onto the same vector.
Carcinogen-induced hepatoma in immunocompetent animal models has shown a progress similar to the clinical course of human hepatoma. Ultrasonography (U/S) was used for consecutive evaluation of the phenotypic changes in Sprague-Dawley rats exposed for 8 weeks to NNM (175 mg/L). Three distinctive US findings were ascites, coarseness (defined as small and
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
S67