- Email: [email protected]
322. hNIS-IRES-GFP Dual Reporter Gene Imaging
CANCER TARGETED GENE THERAPY I 322. hNIS-IRES-GFP Dual Reporter Gene Imaging Jiantu Che, Michael Doubrovin, Joseph Osborne, Inna Serganova, Ludmila Ageyeva, Juri Gelovani (Tjuvajev), Ronald G. Blasberg. 1 Neurology, Memorial Sloan Kettering Cancer Center, New York, NY; 2Radiology, Memorial Sloan Kettering Cancer Center, New York, NY. Objectives: The human (h) and rodent (m/r) sodium-iodide symporters (NIS) have recently been cloned and are being investigated as potential therapeutic (131I-targeted radiotherapy) and reporter genes. We extend this effort by constructing an hNIS - green fluorescent protein (GFP) IRES-linked hybrid reporter gene for both nuclear and optical imaging. Constitutive and inducible reporter gene expression is being used for monitoring tumor growth and response to therapy, and can be used to monitor the expression of endogenous heat-shock protein (HSP) genes in response to stress, radiation therapy and the ancymycin class of drugs. Methods: A retroviral vector (pQHNIG) containing a hNIS-iresGFP dual-reporter gene was constructed with a constitutive CMV promoter and another vector with an inducible hHSP70 promoter is being constructed. Wild-type RG2 cells were transfected (RG2hNISGFP) and used for both in vitro 131I accumulation and FACS assays, and for in vivo 131I gamma camera and 124I PET imaging studies in tumor-bearing mice. Results: Transduced RG2 cells demonstrated GFP fluorescence, accumulated 131I to a 60/1 tissue/medium (T/M) ratio, compared to T/M of 0.8 in wild-type RG2 cells, and were sorted and selected by FACS. Tumors produced by transduced and wild-type RG2 cells were readily differentiated by in vivo gamma camera and less well by fluorescence imaging; clear differentiation was observed within 10 min of 131I injection, and the transduced-to-wild type tumor activity ratio continued to increase over the 60 minute observation period, reaching a ratio of 4.8 and a value of 2.0 %dose/g (tissue sampling values). Conclusions: The hNIS-ires-GFP dual reporter gene is functional and constitutive expression in stable transduced cell lines has been used for optical and nuclear imaging of tumors produced from these cell lines, and provides the opportinity to monitor tumor growth and response to therapy. The dual-reporter can also be coupled to inducible promoters and provides an alternative to our HSV1-tk GFP fusion reporter construct. It also provides the opportunnity for a wider application of molecular imaging by using different radioiostopes of iodide for both gamma camera and PET imaging.
323. Human Plasminogen Kringle 5 Domain, a Novel and Potent Endothelial Cell-Specific Inhibitor for Anti-Angiogenic Gene Therapy of Cancer
therapeutic transgene within a cancer gene therapy strategy. To test this hypothesis, we have developed a K5-expressing retroviral vector and have characterized the angiostatic activity of the de novo produced K5 peptide in vitro. Methods and Results: To assess the endothelial cell inhibitory properties of retrovector-generated K5 peptide, the 363bp human plasminogen K5 domain cDNA - nt1495nt1734 NCBI LocusLink mRNA Source NM_000301 - (InvivoGen, San Diego, CA) was His-tagged at the C terminus and cloned into a bicistronic retroviral vector comprising the enhanced green fluorescent protein reporter gene. Upon transfection of K5 retrovector plasmid into 293GPG retroviral packaging cells, single clones were drug selected and characterized. Anti-His immunoblot analysis on conditioned supernatant collected from K5 retroviral producer clones revealed a major 18kDa protein consistent with the predicted MW of soluble K5. To determine the functionality of K5 upon its secretion, two in vitro angiogenic assays were performed specifically evaluating the ability of K5 to perturb human umbilical vein endothelial cell (HUVEC) migration as well as capillary tube formation. Conditioned media from stably transfected K5 retroviral producer cells is capable of inhibiting HUVEC migration (up to 93 +/- 5%). In addition, the viral supernatants from stably transfected K5 gene-modified producer cells were also capable of preventing the formation of HUVEC-derived capillary tube structures as compared to their null and mock transfected controls. The ability of K5 to antagonize tube structure formation is even more evident upon 18 hours exposure, where complete elimination of capillary tubes was observed (Figure 1).
Conclusions: Our results reveal that K5 gene-modified retroviral producer cells are capable of appropriately secreting K5, and that the K5 domain on its own can serve as a potent angiostatic agent in suppressing endothelial cell migration and capillary tube structure formation in vitro. Our data supports the use of a K5 retroviral gene delivery platform for anti-angiogenic cancer therapy.
324. Retargeting Measles Virus for Therapy of Multiple Myeloma
Sabrina R. Perri,1 Borhane Annabi,2 Richard Béliveau,2 Jacques Galipeau.3 1 Medicine, Lady Davis Institute, McGill University, Montreal, QC, Canada; 2Biochemistry, Université de Québec à Montréal, Montreal, QC, Canada; 3Hematology-Oncology, Lady Davis Institute, Jewish General Hospital, Montreal, QC, Canada.
Kah-Whye Peng,1 Takafumi Nakamura,1 Sompong Vongpunsawad,1 Kathleen A. Donovan,2 John A. Lust,2 Roberto Cattaneo,1 Stephen J. Russell.1 1 Molecular Medicine Program, Mayo Clinic, Rochester, MN; 2 Department of Hematology, Mayo Clinic, Rochester, MN.
Objective: It is now well recognized that tumor angiogenesis plays an essential role in tumor progression. It would thus be highly desirable to exploit the properties of known anti-angiogenic agents to treat cancer. Several potent inhibitors of the angiogenic process are known. Amongst them, angiostatin, is a cleavage product of human plasminogen encompassing the first four N-terminal kringle structures. Of particular interest is the fifth kringle (K5) of plasminogen that has been found to enhance the angiostatic potency of angiostatin. We propose that the K5 domain may serve as a potent angiostatic agent on its own and that it may act as a useful
Multiple myeloma is an incurable disease and innovative therapies are urgently needed. We previously demonstrated that the live attenuated Edmonston B strain of measles virus (MV-Edm) has potent cytopathic activity against primary multiple myeloma cells and against human myeloma xenografts in mice. Since myeloma is a disseminated disease, virotherapy using MV-Edm will require systemic administration of the virus. Two immediate issues to address regarding this route for therapy are inhibition of virus infection due to pre-existing antibodies to measles virus in pre-exposed or immunized individuals and virus infection of non-target tissues. The majority of patients with advanced myeloma have a
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
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323. Human Plasminogen Kringle 5 Domain, a Novel and Potent Endothelial Cell-Specific Inhibitor for Anti-Angiogenic Gene Therapy of Cancer
therapeutic transgene within a cancer gene therapy strategy. To test this hypothesis, we have developed a K5-expressing retroviral vector and have characterized the angiostatic activity of the de novo produced K5 peptide in vitro. Methods and Results: To assess the endothelial cell inhibitory properties of retrovector-generated K5 peptide, the 363bp human plasminogen K5 domain cDNA - nt1495nt1734 NCBI LocusLink mRNA Source NM_000301 - (InvivoGen, San Diego, CA) was His-tagged at the C terminus and cloned into a bicistronic retroviral vector comprising the enhanced green fluorescent protein reporter gene. Upon transfection of K5 retrovector plasmid into 293GPG retroviral packaging cells, single clones were drug selected and characterized. Anti-His immunoblot analysis on conditioned supernatant collected from K5 retroviral producer clones revealed a major 18kDa protein consistent with the predicted MW of soluble K5. To determine the functionality of K5 upon its secretion, two in vitro angiogenic assays were performed specifically evaluating the ability of K5 to perturb human umbilical vein endothelial cell (HUVEC) migration as well as capillary tube formation. Conditioned media from stably transfected K5 retroviral producer cells is capable of inhibiting HUVEC migration (up to 93 +/- 5%). In addition, the viral supernatants from stably transfected K5 gene-modified producer cells were also capable of preventing the formation of HUVEC-derived capillary tube structures as compared to their null and mock transfected controls. The ability of K5 to antagonize tube structure formation is even more evident upon 18 hours exposure, where complete elimination of capillary tubes was observed (Figure 1).
Conclusions: Our results reveal that K5 gene-modified retroviral producer cells are capable of appropriately secreting K5, and that the K5 domain on its own can serve as a potent angiostatic agent in suppressing endothelial cell migration and capillary tube structure formation in vitro. Our data supports the use of a K5 retroviral gene delivery platform for anti-angiogenic cancer therapy.
324. Retargeting Measles Virus for Therapy of Multiple Myeloma
Sabrina R. Perri,1 Borhane Annabi,2 Richard Béliveau,2 Jacques Galipeau.3 1 Medicine, Lady Davis Institute, McGill University, Montreal, QC, Canada; 2Biochemistry, Université de Québec à Montréal, Montreal, QC, Canada; 3Hematology-Oncology, Lady Davis Institute, Jewish General Hospital, Montreal, QC, Canada.
Kah-Whye Peng,1 Takafumi Nakamura,1 Sompong Vongpunsawad,1 Kathleen A. Donovan,2 John A. Lust,2 Roberto Cattaneo,1 Stephen J. Russell.1 1 Molecular Medicine Program, Mayo Clinic, Rochester, MN; 2 Department of Hematology, Mayo Clinic, Rochester, MN.
Objective: It is now well recognized that tumor angiogenesis plays an essential role in tumor progression. It would thus be highly desirable to exploit the properties of known anti-angiogenic agents to treat cancer. Several potent inhibitors of the angiogenic process are known. Amongst them, angiostatin, is a cleavage product of human plasminogen encompassing the first four N-terminal kringle structures. Of particular interest is the fifth kringle (K5) of plasminogen that has been found to enhance the angiostatic potency of angiostatin. We propose that the K5 domain may serve as a potent angiostatic agent on its own and that it may act as a useful
Multiple myeloma is an incurable disease and innovative therapies are urgently needed. We previously demonstrated that the live attenuated Edmonston B strain of measles virus (MV-Edm) has potent cytopathic activity against primary multiple myeloma cells and against human myeloma xenografts in mice. Since myeloma is a disseminated disease, virotherapy using MV-Edm will require systemic administration of the virus. Two immediate issues to address regarding this route for therapy are inhibition of virus infection due to pre-existing antibodies to measles virus in pre-exposed or immunized individuals and virus infection of non-target tissues. The majority of patients with advanced myeloma have a
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
S127