Grant Mcfadden
University Of Florida
Project start date: 2010-03-01
Project end date: 2014-12-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Grant Mcfadden
STUDIES IN POXVIRUS HOST RANGE GENES AND TROPISM
Grant Mcfadden
University Of Florida, 219 Grinter Hall, Gainesville, Fl 32611-5500
Grant 5R01AI080607-02 from National Institute Of Allergy And Infectious Diseases
Abstract: We propose to examine the molecular pathways that mediate the species specificity and tropism of one particular poxvirus, namely myxoma virus (MV). MV is a rabbit-specific poxvirus that induces distinctly different disease profiles depending on the specific rabbit species but is nonpathogenic for every other host species, including man. Our lab has extensively studied MV and the disease it causes in rabbits, called myxomatosis, as a model system to investigate the fundamental principles of poxvirus pathogenesis, particularly by exploiting our large and growing collection of targeted MV gene knockout constructs. Several years ago, we unexpectedly discovered that primary mouse cells, which are normally nonpermissive for MV infection, could be rendered fully permissive by interrupting the cellular interferon (IFN) responses. This work then led to the discovery that the majority of human cancer cells tested were fully permissive for MV and that MV is remarkably effective as oncolytic therapy for the treatment of human cancer xenografts in a variety of animal models. We also discovered that two viral host range factors (M-T5 and M063) were also critical for permissive MV replication in many human cancer cells and we could manipulate viral permissiveness and oncolysis through these viral proteins and their host cell protein targets. Finally, our most recent observation related to this proposal is that primary human cells are protected from MV infection synergistically by IFN and tumor necrosis factor (TNF). We propose to 1- Evaluate the roles of viral host range factors in MV tropism for human cancer cells. We propose to analyze the interactions of MV host range proteins with host cell signaling molecules (like Akt) by mutagenesis, siRNA knockdowns, signaling inhibitors and protein microarrays. We will test a number of adjunct strategies, like signaling modifier drugs used for cancer chemotherapy in man, to increase MV oncolytic potential for a wider spectrum of human cancer cells. 2- Investigate the role of host IFN and TNF responses in MV tropism. We will study MV knockout viruses that are deleted in several key viral host range genes to investigate their roles in inhibiting IFN or TNF responses, and modulating MV tropism and oncolysis. This new information will allow for more rational approaches to optimizing MV virotherapy against a wider spectrum of human cancers, and for controlling MV replication in primary noncancerous human cells. Previously, our studies on poxvirus pathogenesis were more focused on studying the basic mechanisms by which one particular rabbit-specific poxvirus called myxoma virus (MV) causes disease in the rabbit host. Recently, we discovered that MV also infects and kills a wide spectrum of human cancer cells and we have now used MV to successfully treat several types of cancers in animal models. In this proposal, we will study two key viral host range factors and their cellular signaling pathway targets in order to assist the preclinical development of MV as a new oncolytic therapeutic for cancer in man
Keywords: Animal Model; Animal Models and Related Studies; Australia; Benign; Biological Models; CUL-1; Cachectin; Cachectin-Tumor Necrosis Factor; Cancer Control; Cancer Control Science; Cancers; Cell Communication and Signaling; Cell Signaling; Cells; Chemotherapy Protocol; Chemotherapy Regimen; Chemotherapy, Cancer, General; Chemotherapy-Oncologic Procedure; Collection; Combination Chemotherapy Regimen; Complex; Cul-1 protein; Cullin 1; Cullin 1 Protein; Cullin Homolog 1; Development; Disease; Disorder; Drug usage; Drugs; EC 2.7; Embryo; Embryonic; Esocidae; European; Fibroblasts; Gene Targeting; Genes; Genetics-Mutagenesis; Glial Cell Tumors; Glial Neoplasm; Glial Tumor; Glioma; Heterograft; Human; Human, General; IFN; Immunocompetent; Immunodeficient Mouse; Infection; Interferons; Intracellular Communication and Signaling; Killings; Kinases; Knock-out; Knockout; Lagomorpha; Lagomorphs; Libraries; Lin-19 protein; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Mammals, Rabbits; Man (Taxonomy); Man, Modern; Mediating; Medication; Mice; Model System; Models, Biologic; Molecular; Molecular Biology, Mutagenesis; Murine; Mus; Mutagenesis; Myxoma virus; Myxomatosis Virus; Neoplasms of Neuroglia; Neuroglial Neoplasm; Neuroglial Tumor; New Zealand; Oncolytic; Oryctolagus cuniculus; Pathogenesis; Pathway interactions; Permissivenesses; Pharmaceutic Preparations; Pharmaceutical Preparations; Phosphotransferases; Pike; Pike fish; Population; Poxviridae; Poxvirus Myxomatis; Poxviruses; Proliferating; Protein Biochips; Protein Chips; Protein Microarray; Protein Microchips; Proteins; Quimioterapia; RNA, Small Interfering; Rabbit, Domestic; Rabbits; Rapamune; Rapamycin; Reporting; Role; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Molecule; Sirolimus; Small Interfering RNA; Species Specificity; T-Cells; T-Lymphocyte; TNF (unspecified); TNF Receptor Ligands; TNF-alpha; Targetings, Gene; Testing; Therapeutic; Thymus-Dependent Lymphocytes; Transphosphorylases; Transplantation, Heterologous; Tropism; Tumor Necrosis Factor; Tumor Necrosis Factor Family Protein; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Tumors of Neuroglia; Viral; Viral Diseases; Viral Gene Products; Viral Gene Proteins; Viral Proteins; Virus; Virus Diseases; Virus Replication; Viruses, General; Work; Xenograft; Xenograft procedure; Xenotransplantation; analog; biological signal transduction; cancer cell; cancer chemotherapy; cancer type; disease/disorder; drug use; drug/agent; enzootic; feral; gene product; inhibitor; inhibitor/antagonist; knockout gene; malignancy; man; man`s; member; model organism; neoplasm/cancer; novel; oncolysis; pathway; permissiveness; pox virus; pre-clinical; preclinical; protein protein interaction; response; siRNA; skin lesion; social role; thymus derived lymphocyte; tool; tumor; tumor necrosis factor (unspecified); viral infection; virus infection; virus multiplication; virus protein; virus tropism
Relevance: Previously, our studies on poxvirus pathogenesis were more focused on studying the basic mechanisms by which one particular rabbit-specific poxvirus called myxoma virus (MV) causes disease in the rabbit host. Recently, we discovered that MV also infects and kills a wide spectrum of human cancer cells and we have now used MV to successfully treat several types of cancers in animal models. In this proposal, we will study two key viral host range factors and their cellular signaling pathway targets in order to assist the preclinical development of MV as a new oncolytic therapeutic for cancer in man
Project start date: 2009-03-16
Project end date: 2014-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-07-070
5R01AI080607-02 (2010): $358685
NUCLEIC ACIDS PROGRAMMABLE PROTEIN ARRAY CORE FOR PATHOGENIC HUMAN VIRUSES
Grant Mcfadden
University Of North Carolina Chapel Hill, Office Of Sponsored Research, Chapel Hill, Nc 27599
Abstract: This core proposal is to exploit viral pathogen protein microarrays for the purpose of identifying novel hostvirus protein interacting partners that regulate virus-host species tropism, host innate immune response and mediate pathogenic zoonotic infections in man. We propose to utilize a newly developed technology, NAPPA (Nucleic Acid Programmable Protein Arrays), which is currently being developed at the Harvard Proteomic Facility for the human proteome. We are now completing the construction of the monkeypox virus (MPV) proteome array (strain Zaire), but will expand the repertoire of the microarrays to include other viral pathogens of interest to SERCEB and the RCE program starting with, human coronaviruses, dengue and Chikungunya virus. The virus-specific protein microarrays will be used to screen for novel human-virus protein interactions, with emphasis initially on the human inflammasome pathway, the interferon (IFN) pathway, and tumor necrosis factor (TNF) signaling pathway members. Viral proteome microarrays also have utility for studying anti-viral immune responses, screening for novel viral drug targets, and developing novel diagnostic strategies. Through the use of this proteomic platform technology, we seek to address the hypothesis that there are specific protein-protein interactions between proteins from human viral pathogens and human antiviral elements that are critical for host species tropism and pathogenesis in primates and man. By identifying viral/host protein interactions that regulate tropism and disease virulence properties of these viruses, we seek to identify specific targets for the development of novel antiviral strategies, as well as to better define the regulators of viral-induced pathogenic syndromes, such as the virus-induced "cytokine storm" that is thought to underlie the pathogenesis associated with a variety of zoonotic viral diseases in man. This project is being pursued in collaboration with the Harvard Proteomics Facility (Director Josh LaBaer), which is developing the NAPPA technology to create human and select bacterial pathogen protein microarrays
Keywords: Address; Antiviral Agents; Antiviral Drugs; Antivirals; Belgian Congo; Breakbone Fever Virus; Cachectin; Cachectin-Tumor Necrosis Factor; Chikungunya virus; Chimera Protein; Chimeric Proteins; Collaborations; Complex; Congo (Kinshasa); Democratic Republic of the Congo; Dengue; Dengue Fever; Dengue Virus; Development; Disease; Disorder; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Drug Therapy; Egypt 101 virus; Elements; Fusion Protein; Generations; Genome; Goals; Human; Human Virus; Human, General; IFN; Immune response; Immune system; Interferons; Investigators; Libraries; Mammals, Primates; Man (Taxonomy); Man, Modern; Mediating; Microarray Analysis; Microarray-Based Analysis; Molecular; Monkey Pox Virus; Monkeypox virus; Monkeypoxvirus; Nucleic Acids; ORFs; Open Reading Frames; Pathogenesis; Pathway interactions; Pharmacotherapy; Plasmids; Primates; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Protein Array; Protein Binding; Protein Biochips; Protein Chips; Protein Coding Region; Protein Microarray; Protein Microchips; Proteins; Proteome; Proteomics; Research Personnel; Researchers; Screening procedure; Serotyping; Signal Pathway; Syndrome; TNF (unspecified); TNF Receptor Ligands; TNF-alpha; Technology; Tropism; Tumor Necrosis Factor; Tumor Necrosis Factor Family Protein; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Viral; Viral Diseases; Viral Gene Products; Viral Gene Proteins; Viral Proteins; Virulence; Virus; Virus Diseases; Viruses, General; WNV; West Nile; West Nile virus; Zaire; Zoonotic Infection; base; biodefense; body system, allergic/immunologic; breakbone fever; cytokine; disease/disorder; expression vector; gene product; host response; human coronavirus; immunoresponse; interest; man; man`s; member; microarray technology; new diagnostics; next generation diagnostics; novel; novel diagnostics; organ system, allergic/immunologic; pathogen; pathway; programs; protein protein interaction; response; screening; screenings; tumor necrosis factor (unspecified); viral infection; virus infection; virus protein
Relevance: This project will screen a diverse variety of viral pathogens of man for viral proteins that interact with the human innate immune system. This analysis will uncover new targets for drug therapy that can potentially act against diverse viral pathogens such as monkeypox virus, dengue, and human coronaviruses like SARS, as well as new emerging diseases like Chikungunya virus
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
5U54AI057157-08_5834 (2010): $202050
MYXOMA VIRUS (MV) ONCOLYSIS FOR TREATING HUMAN CANCER
Grant Mcfadden
University Of Florida, 219 Grinter Hall, Gainesville, Fl 32611-5500
Grant 1R01CA138541-01A2 from National Cancer Institute
Abstract: Myxoma virus (MV) infects only rabbits in vivo, but also has a natural capacity to infect a wide variety of human cancer cells in vitro and in vivo. Thus, MV is an attractive candidate for oncolytic virotherapy to treat human cancer. MV has been used to successfully treat several diverse human brain cancers in xenografted immunodeficient mice and murine metastatic melanoma in immunocompetent mice. Here, MV will be developed for human clinical trials by exploiting a therapeutic strategy for which the virus is uniquely well-suited ex vivo purging of cancer cells within human bone marrow or mobilized-PBMC samples from patients who would normally be excluded from autologous stem cell transplantation following high dose chemotherapy. Considerable preliminary data has been collected to support this proposal 1) MV does not perturb or compromise human multipotent stem cell differentiation in immunodeficient mice engrafted with normal human bone marrow or cytokine-mobilized PBMCs, 2) MV eliminates a wide variety of human cancer cells following ex vivo purging, 3) MV can effectively purge not only permissive leukemia/lymphoma cells in vivo, but also unexpectedly prevents even nonpermissive human leukemia cells (such as KG1 cells) from engraftment or tumor induction, and 4) MV recombinants that express a variety of useful reporter proteins (fluorescent and bioluminescent) for imaging purposes have already been constructed. Specifically, our aims are 1) Validate MV safety for ex vivo treatment of normal human hematopoietic stem cells The safety of MV-purging for normal human stem cell differentiation will be tested, using engrafted immunodeficient NOG mice to verify full hematopoietic cell engraftment and immune reconstitution. MV purging will be tested on primary human stem/progenitor cells derived from normal bone marrow and G-CSF-mobilized PBMCs, using hematopoietic colony forming cell assays in vitro as well as for efficient hematologic cell engraftment in vivo. 2) Optimize MV ex vivo cancer cell purging Two human cancers, B-cell lymphoma and acute myeloid leukemia, will be investigated for the ability of ex vivo MV purging to eliminate their tumorigenic potential in vivo in engrafted NOG mice. The cancer cells and viruses will be tagged with distinguishable luciferases that allow the engrafted tumor cells and the therapeutic virus to be independently tracked in vivo. Primary cells from acute myeloid leukemia patients will also be tested for the ability of MV to specifically eliminate the contaminating cancer cells and allow the selective engraftment of only noncancerous human leukocytes. 3) Investigate the mechanism of MV purging of primary human leukemia cells We have recently shown that ex vivo infection of human KG1 leukemia cells with MV prevents the subsequent engraftment and tumor formation of these cells into NOG recipient mice, despite the fact that these cells are completely nonpermissive for MV infection in vitro. To assess for virus-induced cell signaling changes, we probed MV-infected KG1 cells with an array of antibodies to 46 different human signaling phosphoproteins, and observed that MV infection specifically induces Stat5 and Hck activation in KG1 cells. We will explore the functional significance of these host cell signaling activations for the successful ex vivo tumor cell purging of human leukemia calls by MV. PUBLIC HEALTH RELEVANCE Recently, we discovered that one particular rabbit-specific poxvirus, called myxoma virus (MV), also infects and kills a wide spectrum of human cancer cells and MV has been used to successfully treat several types of cancers in animal models. In order to facilitate the preclinical development of MV as a new oncolytic therapeutic for cancer in man, we propose to exploit two specific cancer models (human B-cell lymphoma and acute myeloid leukemia) to establish the optimal conditions for a novel therapeutic "cancer cell purging" protocol. This strategy will benefit leukemia/lymphoma cancer patients who are currently excluded from autologous bone marrow transplantation by allowing their own stem cell samples (from bone marrow or blood) to be purged of cancer cells prior to re-engraftment and reconstitution of their immune system following high dose chemotherapy
Keywords: ABMT; AML - Acute Myeloid Leukemia; Animal Model; Animal Models and Related Studies; Antibodies; Autologous Bone Marrow Transplant; Autologous Bone Marrow Transplantation; Autologous Marrow Transplantation; Autologous Stem Cell Transplantation; B lymphoma; B-Cell Lymphomas; B-Cell Non-Hodgkin`s Lymphoma; B-Cell NonHodgkins Lymphoma; Blood; Blood (Leukemia); Blood Precursor Cell; Blood leukocyte; Body Tissues; Bone Marrow; Cancer Model; Cancer Patient; Cancer cell line; Cancer of Brain; CancerModel; Cancers; Cell Communication and Signaling; Cell Signaling; Cells; Clinic; Clinical Trials; Clinical Trials, Unspecified; Data; Development; Drugs; Engraftment; Florida; Hematopoietic; Hematopoietic stem cells; Heterograft; High Dose Chemotherapy; Human; Human, General; Image; Immune; Immune system; Immunocompetent; Immunodeficient Mouse; Immunologic, Luciferase; In Vitro; Infection; Injection of therapeutic agent; Injections; Intracellular Communication and Signaling; Killings; Leukemia, Myelocytic, Acute; Leukemias, General; Leukocytes; Libraries; Luciferases; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Malignant Tumor of the Brain; Malignant neoplasm of brain; Mammals, Mice; Mammals, Rabbits; Man (Taxonomy); Man, Modern; Marrow leukocyte; Medication; Metastatic Melanoma; Mice; Molecular; Mother Cells; Multipotent Stem Cells; Murine; Mus; Myeloblastic Leukemia, Acute; Myelogenous Leukemia, Acute; Myxoma virus; Myxomatosis Virus; Normal Tissue; Normal tissue morphology; Oncolytic; Oryctolagus cuniculus; PBSC; Patients; Peripheral Blood Stem Cell; Peripheral Stem Cells; Pharmaceutic Preparations; Pharmaceutical Preparations; Phosphoproteins; Poxviridae; Poxvirus Myxomatis; Poxviruses; Progenitor Cells; Progenitor Cells, Hematopoietic; Proteins; Protocol; Protocols documentation; Rabbit, Domestic; Rabbits; Rapamune; Rapamycin; Receptors, Virus; Recombinants; Refractory; Reporter; Reticuloendothelial System, Blood; Reticuloendothelial System, Bone Marrow; Reticuloendothelial System, Leukocytes; Safety; Sampling; Signal Transduction; Signal Transduction Systems; Signaling; Sirolimus; Source; Stem cells; Testing; Therapeutic; Tissues; Transplantation; Transplantation, Heterologous; Tropism; Tumor Cell; Viral Diseases; Viral Genetics; Viral Receptor; Virus; Virus Diseases; Virus Receptors; Viruses, General; White Blood Cells; White Cell; Xenograft; Xenograft procedure; Xenotransplantation; acute granulocytic leukemia; acute myeloid leukemia; acute nonlymphocytic leukemia; base; biological signal transduction; body system, allergic/immunologic; cancer cell; cancer type; clinical investigation; cytokine; drug/agent; gene product; human stem cells; imaging; in vitro Assay; in vivo; leukemia; leukemia/lymphoma; lymphoma/leukemia; malignancy; man; man`s; model organism; multipotent progenitor; mutant; neoplasm/cancer; neoplastic cell; new therapeutics; next generation; next generation therapeutics; novel therapeutics; oncolysis; organ system, allergic/immunologic; pox virus; pre-clinical; preclinical; prevent; preventing; public health relevance; purge; purging; recombinant virus; reconstitute; reconstitution; response; stem; stem cell differentiation; therapeutic transgene; transplant; tumor; tumorigenic; viral infection; virus genetics; virus infection; virus tropism; white blood cell; white blood corpuscle
Relevance: Recently, we discovered that one particular rabbit-specific poxvirus, called myxoma virus (MV), also infects and kills a wide spectrum of human cancer cells and MV has been used to successfully treat several types of cancers in animal models. In order to facilitate the preclinical development of MV as a new oncolytic therapeutic for cancer in man, we propose to exploit two specific cancer models (human B-cell lymphoma and acute myeloid leukemia) to establish the optimal conditions for a novel therapeutic "cancer cell purging" protocol. This strategy will benefit leukemia/lymphoma cancer patients who are currently excluded from autologous bone marrow transplantation by allowing their own stem cell samples (from bone marrow or blood) to be purged of cancer cells prior to re-engraftment and reconstitution of their immune system following high dose chemotherapy
Project start date: 2010-03-01
Project end date: 2014-12-31
Budget start date: 1-MAR-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
1R01CA138541-01A2 (2010): $303988
Grant Mcfadden
University Of Florida
Project start date: 2011-01-01
Project end date: 2012-12-31