- Email: [email protected]
533. Examining the Role of CD44 in the Tumor Tropism of Multipotent Mesenchymal Stem Cell (MSC) for Cancer Microenvironments
CANCER-TARGETED GENE & CELL THERAPY II The c-Met receptor can interact with multiple intracellular signaling pathways, and is up regulated in a majority of malignancies. This makes c-Met a relevant target for novel therapeutic interventions. Virotherapy employing oncolytic adenoviruses represents a promising biological intervention applicable to a wide array of neoplastic diseases. Using oncolytic adenoviruses, designed to divide in and specifically kill tumor cells, is a promising virotherapy approach for cancer treatment. Ideally, cancer-specific replication of oncolytic adenoviruses results in viral-mediated replication and lysis of infected tumor tissues. Release of virus progeny results in further propagation in surrounding tumor cells but not in those of normal tissues that would be refractory to virus replication. To date, achieving replicative specificity of oncolytic adenovirus agents has been accomplished primarily by incorporating tumor selective promoters. To achieve a novel alternative specificity of oncolytic adenoviruses, we used a high affinity c-Met ligand to retarget viral infectivity to metastatic disease over expressing the receptor. Specifically, we incorporated NK2, a competitive antagonist of the HGF - c-Met association, into the adenovirus fiber. In this approach, the NK2 ligand was genetically incorporated by ablating the adenovirus fiber (protein pIV) gene, and replacing it with a recombinant gene consisting of the tail domain from the fiber and a trimerization domain from T4 fibritin fused to NK2 (fiber-fibritin-NK2). This study demonstrates the ability to rescue viable adenoviral particles that display functional fiber-fibritin-NK2 as a component of their capsid surface. Importantly, this construct in the context of an oncolytic adenovirus, specifically infected cancer cell lines that over express c-Met, and induced cell lysis. These results suggest that a c-Met retargeted virus could be used to counteract tumor mitogenesis, motogenesis, and morphogenesis driven by this receptor. If successful, this approach could open new avenues for using adenoviral vectors in cancer gene therapy and hasten the shift from preclinical to clinical trials.
Cancer-Targeted Gene & Cell Therapy II 531. Stanniocalcin-1 Derived from Multi Potent Stromal Cell Changes Cell Metabolism Dramatically, Decreases Reactive Oxygen Species Stress and Promotes Survival of Cancer Cells with Uncoupling Protein 2 Upregulation
Shinya Ohkouchi,1 Gregory J. Block,2 Darwin J. Prockop,3 Toshihiro Nukiwa.1 1 Department of Respiratory Medicine, Graduate School of Medicine, Tohoku University, Sendai, Miyagi, Japan; 2Institute of Sten Cell and Regenerative Medicine, University of Washington, Seattle, WA; 3Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Temple, TX.
Previous studies have demonstrated that multipotent stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1) in lactic acidosis and hypoxia. These situation induce lots of intracellular ROS, therefore we hypothesized MSCs derived STC1 decreases ROS induced cell death. This study shows that MSC derived STC1 promotes survival of lung cancer A549 cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more uncoupling metabolic profile. MSC derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549 cells in an STC1 dependent manner. Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population. rSTC1 treated A549 cells displayed decreased levels of reactive oxygen species, mitochondrial membrane potential, increased lactate production and increased oxygen consumption, all of which were dependent on the upregulation of UCP2. Our data suggest that S204
MSCs promote cell survival by regulating mitochondrial respiration via STC1. Furthermore, STC1 may provide promising avenues for treatment of reactive oxygen species and metabolic disorders.
532. Neuropilin-1 Expressing Mononuclear Cells (NEMs), a Novel Population of Bone Marrow Cells Recruited by AAV2-Sema3A, Contributes to Vessel Normalization and Inhibits Tumor Growth Serena Zacchigna,1 Alessandro Carrer,1 Silvia Moimas,1 Lorena Zentilin,1 Mauro Giacca.1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology - ICGEB, Trieste, Italy.
Over the last several years, we have extensively exploited the properties of viral vectors based on the Adeno-Associated Virus (AAV) for the in vivo delivery of a variety of genes involved in blood vessel formation in various small and large animal models. AAV vectors have the capacity to transduce non-replicating cells with high efficiency, in the absence of inflammation and immune response, and thus promote expression of their transgenes in vivo for indefinite periods of time. In particular, we observed that the prolonged expression of VEGF-A165 and Sema3A, two Neuropilin-1 (Nrp-1) ligands, determined a massive infiltration of the expressing tissues by a population of bone-marrow derived mononuclear cells expressing Nrp-1. These Nrp-1 Expressing Mononuclear cells (NEMs) were found to be involved in vessel maturation through a paracrine effect ensuing in the activation and proliferation of tissue-resident mural cells (Zacchigna et al. J. Clin. Invest. 2008, 118, 2062). Consistent with the observation that NEMs quench angiogenic burden and promote vessel maturation, we observed that both AAV-Sema3A and NEMs exerted strong anti-tumoral effect. In particular, we exploited AAV2-mediated gene transfer to overexpress Sema3A at the site of inoculation of either B16.F10 melanoma or T24.1 fibrosarcoma cells in immunocompetent mice. We found that Sema3A markedly reduced tumor growth, without directly affecting tumor cell proliferation. Similarly, the direct injection of NEMs, isolated from either the bone marrow or AAV-Sema3A-injected muscles, and administered to B16-F10 tumor-bearing animals, markedly impaired tumor growth. Analysis of the vasculature of AAV2-Sema3A- or NEMtreated tumors revealed a more mature vascular network, associated to a higher degree of a-SMA+/NG2+ mural cell coverage, reduced vessel tortuosity and decreased vascular leakiness, indicating a role for NEMs in triggering vessel normalization. Tumors treated with Sema3A or NEMs were smaller, better perfused, less hypoxic and with a reduced level of activation of HIF-1a. Phenotypic characterization of purified NEMs revealed that they constitute a unique population of cells, having a CD11b+/Nrp1+/Gr1-/Tie2- phenotype. These cells constitute ∼1.0% of total bone marrow cells and are characterized by a gene expression profile reminiscent of M1 polarized monocyte/ macrophages.
533. Examining the Role of CD44 in the Tumor Tropism of Multipotent Mesenchymal Stem Cell (MSC) for Cancer Microenvironments
Erika L. Spaeth,1 Brian Toole,2 Ann Klopp,1 Michael Andreeff,1 Frank C. Marini.1 1 Leukemia, MD Anderson Cancer Center, Houston, TX; 2Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC.
Multipotent mesenchymal stem/stromal cells (MSC) make excellent gene delivery vehicles because they are readily propagated in vitro, can be genetically modified, have high metabolic activity to support the production of therapeutic agents and have tumor tropic properties. We have shown the propensity of MSC to migrate to and incorporate into the tumor stroma. Once engrafted in the tumor, MSC are capable Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
CANCER-TARGETED GENE & CELL THERAPY II of providing extracellular matrices, cytokines, growth factors and vascular networks for nutrient and waste exchange. We modulate the receptor surface expression of CD44 on MSC to alter the migration of towards tumor conditioned media in vitro and engrafted tumors in vivo. Blocking MSC-expressed CD44 with neutralizing antibody, soluble CD44 decoy receptor, or shRNA knockdown inhibited the migration of MSC toward tumor conditioned media in vitro by greater than 75%, 82% and 50%, respectively. In vivo, 60% inhibition of systemically injected MSC towards bilateral orthotopic engrafted xenograft tumors was achieved using a soluble CD44 decoy receptor. Additionally, using a CD44 luciferase promoter construct, we found that CD44 mRNA expression in MSC varies in response to stimulation by various tumor cell lines. Also, real time PCR analysis showed that tumor stimulated MSC alter their CD44 splice variant expression suggesting alternative variants may enhance migration. These results suggest that CD44 is significant to the tumor-tropic migration capacity of MSC. Furthermore, enhancing the surface expression of CD44 may heighten tumor-tropic migration thereby increasing the efficacy of the MSC as a potential drug delivery vehicle.
Our experiments indicate an effective down-regulation of survivin, a cell protective protein up-regulated in tumor cells. This was accomplished by the modification of a non-toxic but ineffective PEI with different hydrophobic moieties. The resultant polymers showed a significant capability for cellular delivery of siRNA, which in turn was translated into apoptosis induction by CA-substituted PEIs. This study introduces a promising delivery system for safe and effective siRNA delivery, which will be further investigated in preclinical animal models.
535.
Bacterial Cell Therapy for Cancer
Hamidreza Montazeri Aliabadi,1 Breanne Landry,1 Parvin Mahdipoor,1 Hasan Uludag.1 1 Department of Chemical & Material Engineering, University of Alberta, Edmonton, AB, Canada.
Michelle Cronin,1 Sara A. Collins,1,2 Chwanrow Baban,1 Akihito Inagaki,2 Kei Hiraoka,2 Noriyuki Kasahara,2 Kevin Francis,3 Ali Akin,3 Cormac G. M. Gahan,4 Gerald C. O’Sullivan,1 Mark Tangney.1,2 1 Cork Cancer Reserarch Centre, University College Cork, Cork, Ireland; 2David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 3Caliper Life Sciences, Alameda, CA; 4Alimentary Pharmabiotic Centre, Microbiology Dept., School of Pharmacy, University College Cork, Cork, Ireland.
Post-transcriptional silencing of ‘aberrant’ genes is a promising strategy for cancer therapy, and RNA interference (RNAi) has been recognized as a mechanism involved in this silencing. Delivering siRNA to intracellular targets, however, has been proven to be quite a challenge due to inability of anionic siRNA to cross cellular membranes. High molecular weight polyethylenimines (PEIs) are effective siRNA delivery agents; however, their toxicity has been an important obstacle for their clinical use. As an alternative, hydrophobic substitution on small molecular weight PEI with lipophilic moieties has been investigated as a promising approach for effective but non-toxic siRNA carriers. In this study, we report the hydrophobic modification of low molecular PEI with different lipophilic substituents for siRNA delivery to target survivin protein. Survivin is a member of IAP family and has been implicated in multiple essential functions, including cell division and apoptosis. Survivin is an attractive target for anti-cancer therapy, due to (a) low expression in most normal cells and upregulation in malignant tissue, (b) involvement in multiple signalling mechanisms controlling tumor maintenance, and (c) silencing survivin may block angiogenesis and tumor cell growth. We evaluated toxicity of a library of lipidsubstituted PEIs, siRNA uptake, and survivin silencing in MDAMB-231 breast cancer cells using flow cytometry, FlowTACSTM Apoptosis Detection Kit, nuclear staining, and MTT assay. Lipid substitution increased the toxicity of PEI2 for some of the higher substitutions; however, this toxic effect was significantly less than the cytotoxicity displayed by the PEI25 (>50% cell viability for all lipidsubstituted polymers at 10 µg/mL). A significant increase in siRNA cellular uptake was observed when the siRNAs were delivered with lipid-substituted polymers, and significant down-regulation in the target protein expression was established by Caprylic Acid-substituted polymers. The survivin down-regulation was also confirmed by an increase in apoptosis rate and MTT assay to demonstrate a drop in cell viability after polymer/siRNA treatment. Polymer/survivin siRNA treatment prior to anticancer therapy decreased the IC50 of the evaluated drugs upto 49.31-fold.
The prime obstacle to achieving an effective treatment for cancer is that of eradicating tumours without harming healthy cells. Use of biological agents for delivery of therapeutic genes to patients has shown great promise. Bacteria present an attractive class of gene vector, possessing a natural ability to grow specifically within tumours following systemic administration. Pathogenic genera (Clostridium and Salmonella) have been examined in clinical trials. However, as disease-causing bugs, their inherent toxicity has outweighed therapeutic responses. Furthermore, the biology of bacterial tumour targeting is poorly understood. We aim to address these failings through technological development of non-pathogenic bacterial strategies. Lactic Acid Bacteria (LAB) have been utilised in food fermentations for centuries, and are ingested regularly by humans. Certain strains are known to colonise the gut (commensal bacteria), living symbiotically with humans. We have engineered a number of LAB and other commensal bacteria (Bifidobacterium, Lactococcus and E.coli) to express heterologous genes within tumours (external to tumour cells) following systemic administration. Luminescent reporter gene (bacterial lux) tagging of the various bacteria permitted real-time visualization of vector, in subcutaneous and orthotopic murine models (melanoma, breast, lung, glioma). Long-term persistence of these replication-competent vectors was observed in tumours (2-6 weeks) in both athymic and immunocompetent mice. Lower bacterial numbers were observed in immunocpmetent animals and low level, delayed anti-vector immune responses (Th1 and Th2) were observed in tumours. Use of 3D luminescence imaging (IVIS) of lux-expressing bacteria enabled characterisation of intratumoural distribution patterns, and co-registration of bacterial lux and tumour derived firefly luciferase revealed an inverse relationship between tumour viability and bacterial load, even in the case of facultatively anaerobic strains. We have engineered strains to secrete heterologous proteins to high levels and these replication competent bacteria can mediate long-term production of soluble agents within tumour masses, presenting a powerful and safe approach to specific gene/cell therapy of primary tumours and secondary metastases.
534. Survivin Downregulation in Human Breast Cancer Cell Line by siRNA Delivery Using Hydrophobically Modified Aliphatic Polymers
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
S205
Cancer-Targeted Gene & Cell Therapy II 531. Stanniocalcin-1 Derived from Multi Potent Stromal Cell Changes Cell Metabolism Dramatically, Decreases Reactive Oxygen Species Stress and Promotes Survival of Cancer Cells with Uncoupling Protein 2 Upregulation
Shinya Ohkouchi,1 Gregory J. Block,2 Darwin J. Prockop,3 Toshihiro Nukiwa.1 1 Department of Respiratory Medicine, Graduate School of Medicine, Tohoku University, Sendai, Miyagi, Japan; 2Institute of Sten Cell and Regenerative Medicine, University of Washington, Seattle, WA; 3Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Temple, TX.
Previous studies have demonstrated that multipotent stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1) in lactic acidosis and hypoxia. These situation induce lots of intracellular ROS, therefore we hypothesized MSCs derived STC1 decreases ROS induced cell death. This study shows that MSC derived STC1 promotes survival of lung cancer A549 cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more uncoupling metabolic profile. MSC derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549 cells in an STC1 dependent manner. Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population. rSTC1 treated A549 cells displayed decreased levels of reactive oxygen species, mitochondrial membrane potential, increased lactate production and increased oxygen consumption, all of which were dependent on the upregulation of UCP2. Our data suggest that S204
MSCs promote cell survival by regulating mitochondrial respiration via STC1. Furthermore, STC1 may provide promising avenues for treatment of reactive oxygen species and metabolic disorders.
532. Neuropilin-1 Expressing Mononuclear Cells (NEMs), a Novel Population of Bone Marrow Cells Recruited by AAV2-Sema3A, Contributes to Vessel Normalization and Inhibits Tumor Growth Serena Zacchigna,1 Alessandro Carrer,1 Silvia Moimas,1 Lorena Zentilin,1 Mauro Giacca.1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology - ICGEB, Trieste, Italy.
Over the last several years, we have extensively exploited the properties of viral vectors based on the Adeno-Associated Virus (AAV) for the in vivo delivery of a variety of genes involved in blood vessel formation in various small and large animal models. AAV vectors have the capacity to transduce non-replicating cells with high efficiency, in the absence of inflammation and immune response, and thus promote expression of their transgenes in vivo for indefinite periods of time. In particular, we observed that the prolonged expression of VEGF-A165 and Sema3A, two Neuropilin-1 (Nrp-1) ligands, determined a massive infiltration of the expressing tissues by a population of bone-marrow derived mononuclear cells expressing Nrp-1. These Nrp-1 Expressing Mononuclear cells (NEMs) were found to be involved in vessel maturation through a paracrine effect ensuing in the activation and proliferation of tissue-resident mural cells (Zacchigna et al. J. Clin. Invest. 2008, 118, 2062). Consistent with the observation that NEMs quench angiogenic burden and promote vessel maturation, we observed that both AAV-Sema3A and NEMs exerted strong anti-tumoral effect. In particular, we exploited AAV2-mediated gene transfer to overexpress Sema3A at the site of inoculation of either B16.F10 melanoma or T24.1 fibrosarcoma cells in immunocompetent mice. We found that Sema3A markedly reduced tumor growth, without directly affecting tumor cell proliferation. Similarly, the direct injection of NEMs, isolated from either the bone marrow or AAV-Sema3A-injected muscles, and administered to B16-F10 tumor-bearing animals, markedly impaired tumor growth. Analysis of the vasculature of AAV2-Sema3A- or NEMtreated tumors revealed a more mature vascular network, associated to a higher degree of a-SMA+/NG2+ mural cell coverage, reduced vessel tortuosity and decreased vascular leakiness, indicating a role for NEMs in triggering vessel normalization. Tumors treated with Sema3A or NEMs were smaller, better perfused, less hypoxic and with a reduced level of activation of HIF-1a. Phenotypic characterization of purified NEMs revealed that they constitute a unique population of cells, having a CD11b+/Nrp1+/Gr1-/Tie2- phenotype. These cells constitute ∼1.0% of total bone marrow cells and are characterized by a gene expression profile reminiscent of M1 polarized monocyte/ macrophages.
533. Examining the Role of CD44 in the Tumor Tropism of Multipotent Mesenchymal Stem Cell (MSC) for Cancer Microenvironments
Erika L. Spaeth,1 Brian Toole,2 Ann Klopp,1 Michael Andreeff,1 Frank C. Marini.1 1 Leukemia, MD Anderson Cancer Center, Houston, TX; 2Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC.
Multipotent mesenchymal stem/stromal cells (MSC) make excellent gene delivery vehicles because they are readily propagated in vitro, can be genetically modified, have high metabolic activity to support the production of therapeutic agents and have tumor tropic properties. We have shown the propensity of MSC to migrate to and incorporate into the tumor stroma. Once engrafted in the tumor, MSC are capable Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
CANCER-TARGETED GENE & CELL THERAPY II of providing extracellular matrices, cytokines, growth factors and vascular networks for nutrient and waste exchange. We modulate the receptor surface expression of CD44 on MSC to alter the migration of towards tumor conditioned media in vitro and engrafted tumors in vivo. Blocking MSC-expressed CD44 with neutralizing antibody, soluble CD44 decoy receptor, or shRNA knockdown inhibited the migration of MSC toward tumor conditioned media in vitro by greater than 75%, 82% and 50%, respectively. In vivo, 60% inhibition of systemically injected MSC towards bilateral orthotopic engrafted xenograft tumors was achieved using a soluble CD44 decoy receptor. Additionally, using a CD44 luciferase promoter construct, we found that CD44 mRNA expression in MSC varies in response to stimulation by various tumor cell lines. Also, real time PCR analysis showed that tumor stimulated MSC alter their CD44 splice variant expression suggesting alternative variants may enhance migration. These results suggest that CD44 is significant to the tumor-tropic migration capacity of MSC. Furthermore, enhancing the surface expression of CD44 may heighten tumor-tropic migration thereby increasing the efficacy of the MSC as a potential drug delivery vehicle.
Our experiments indicate an effective down-regulation of survivin, a cell protective protein up-regulated in tumor cells. This was accomplished by the modification of a non-toxic but ineffective PEI with different hydrophobic moieties. The resultant polymers showed a significant capability for cellular delivery of siRNA, which in turn was translated into apoptosis induction by CA-substituted PEIs. This study introduces a promising delivery system for safe and effective siRNA delivery, which will be further investigated in preclinical animal models.
535.
Bacterial Cell Therapy for Cancer
Hamidreza Montazeri Aliabadi,1 Breanne Landry,1 Parvin Mahdipoor,1 Hasan Uludag.1 1 Department of Chemical & Material Engineering, University of Alberta, Edmonton, AB, Canada.
Michelle Cronin,1 Sara A. Collins,1,2 Chwanrow Baban,1 Akihito Inagaki,2 Kei Hiraoka,2 Noriyuki Kasahara,2 Kevin Francis,3 Ali Akin,3 Cormac G. M. Gahan,4 Gerald C. O’Sullivan,1 Mark Tangney.1,2 1 Cork Cancer Reserarch Centre, University College Cork, Cork, Ireland; 2David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA; 3Caliper Life Sciences, Alameda, CA; 4Alimentary Pharmabiotic Centre, Microbiology Dept., School of Pharmacy, University College Cork, Cork, Ireland.
Post-transcriptional silencing of ‘aberrant’ genes is a promising strategy for cancer therapy, and RNA interference (RNAi) has been recognized as a mechanism involved in this silencing. Delivering siRNA to intracellular targets, however, has been proven to be quite a challenge due to inability of anionic siRNA to cross cellular membranes. High molecular weight polyethylenimines (PEIs) are effective siRNA delivery agents; however, their toxicity has been an important obstacle for their clinical use. As an alternative, hydrophobic substitution on small molecular weight PEI with lipophilic moieties has been investigated as a promising approach for effective but non-toxic siRNA carriers. In this study, we report the hydrophobic modification of low molecular PEI with different lipophilic substituents for siRNA delivery to target survivin protein. Survivin is a member of IAP family and has been implicated in multiple essential functions, including cell division and apoptosis. Survivin is an attractive target for anti-cancer therapy, due to (a) low expression in most normal cells and upregulation in malignant tissue, (b) involvement in multiple signalling mechanisms controlling tumor maintenance, and (c) silencing survivin may block angiogenesis and tumor cell growth. We evaluated toxicity of a library of lipidsubstituted PEIs, siRNA uptake, and survivin silencing in MDAMB-231 breast cancer cells using flow cytometry, FlowTACSTM Apoptosis Detection Kit, nuclear staining, and MTT assay. Lipid substitution increased the toxicity of PEI2 for some of the higher substitutions; however, this toxic effect was significantly less than the cytotoxicity displayed by the PEI25 (>50% cell viability for all lipidsubstituted polymers at 10 µg/mL). A significant increase in siRNA cellular uptake was observed when the siRNAs were delivered with lipid-substituted polymers, and significant down-regulation in the target protein expression was established by Caprylic Acid-substituted polymers. The survivin down-regulation was also confirmed by an increase in apoptosis rate and MTT assay to demonstrate a drop in cell viability after polymer/siRNA treatment. Polymer/survivin siRNA treatment prior to anticancer therapy decreased the IC50 of the evaluated drugs upto 49.31-fold.
The prime obstacle to achieving an effective treatment for cancer is that of eradicating tumours without harming healthy cells. Use of biological agents for delivery of therapeutic genes to patients has shown great promise. Bacteria present an attractive class of gene vector, possessing a natural ability to grow specifically within tumours following systemic administration. Pathogenic genera (Clostridium and Salmonella) have been examined in clinical trials. However, as disease-causing bugs, their inherent toxicity has outweighed therapeutic responses. Furthermore, the biology of bacterial tumour targeting is poorly understood. We aim to address these failings through technological development of non-pathogenic bacterial strategies. Lactic Acid Bacteria (LAB) have been utilised in food fermentations for centuries, and are ingested regularly by humans. Certain strains are known to colonise the gut (commensal bacteria), living symbiotically with humans. We have engineered a number of LAB and other commensal bacteria (Bifidobacterium, Lactococcus and E.coli) to express heterologous genes within tumours (external to tumour cells) following systemic administration. Luminescent reporter gene (bacterial lux) tagging of the various bacteria permitted real-time visualization of vector, in subcutaneous and orthotopic murine models (melanoma, breast, lung, glioma). Long-term persistence of these replication-competent vectors was observed in tumours (2-6 weeks) in both athymic and immunocompetent mice. Lower bacterial numbers were observed in immunocpmetent animals and low level, delayed anti-vector immune responses (Th1 and Th2) were observed in tumours. Use of 3D luminescence imaging (IVIS) of lux-expressing bacteria enabled characterisation of intratumoural distribution patterns, and co-registration of bacterial lux and tumour derived firefly luciferase revealed an inverse relationship between tumour viability and bacterial load, even in the case of facultatively anaerobic strains. We have engineered strains to secrete heterologous proteins to high levels and these replication competent bacteria can mediate long-term production of soluble agents within tumour masses, presenting a powerful and safe approach to specific gene/cell therapy of primary tumours and secondary metastases.
534. Survivin Downregulation in Human Breast Cancer Cell Line by siRNA Delivery Using Hydrophobically Modified Aliphatic Polymers
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
S205