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Novel Breast Cancer Therapeutic Vectors Based on a Paramyxovirus Fusion Protein
Novel Breast Cancer Therapeutic Vectors based on a Paramyxovirus Fusion Protein
This is a resubmission of a proposal that was favorably reviewed last year for funding to create novel viral vectors for breast cancer therapy. We have revised the application in light of the reviewers comments.
Viruses and virus-based vectors represent powerful therapeutic tools. However, a widely recognized limitation of current delivery systems has been the lack of success in controlling the ability of recombinant viruses to kill only predetermined cell types or tissues. This proposal seeks funding to develop novel vectors for breast cancer therapy that are based on controlling cell killing through alterations to a viral fusion protein.
An innovative aspect of our work is the focus on the paramyxovirus Simian Virus 5 (SV5) as a novel therapeutic vector. Among paramyxoviruses, SV5 has great potential as an RNA virus vector, because the virus can infect humans and non-human primates, but is not associated with known disease. Our published work has provided “proof of principle” that SV5 can be engineered for controlled cell killing. Here we propose a mechanism to achieve controlled killing of metastatic tumor cells but not normal cells.
Viral fusogenic membrane proteins are a new class of therapeutic molecules that have been used to promote cell-cell fusion for local control of tumor growth. Among paramyxovirus F proteins, the SV5 F protein is the best candidate to potentially engineer for controlled cell-cell fusion, since fusion occurs at neutral pH, is extremely efficient, and is catalyzed by a single protein with a relatively simple structure. The SV5 F protein has inherent properties that we will exploit to create novel therapeutics for breast cancer, including: 1) SV5 F promotes cell-cell fusion without its cognate attachment protein HN, a property not found with any other F protein, 2) SV5 F is cleavage activated by cellular proteinases, providing a mechanism to control fusion with new cleavage sites, 3) well defined variants of SV5 F have already been developed that have altered protease-activation sites, 4) syncytia formation is the only known cytopathic effect of SV5 infection, raising the possibility of controlling cell killing by controlling F protein activity.
The overall objective of the proposed work is to develop the paramyxovirus SV5 as a therapeutic vector for controlled killing of breast cancer cells. We will test the hypothesis that a rSV5 expressing hyper-fusogenic variants of F will be more efficient at killing breast cancer cells than rSV5 expressing WT F protein. Secondly, we will test the hypothesis that fusion activity and cell killing by an rSV5 vector can be controlled by engineering the SV5 F protein to have a cleavage site for matrix metalloproteinases (MMPs) that are overexpressed in metastatic breast cancer cells.
In aim 1, recombinant SV5 encoding previously identified hyper-fusogenic F variants will be constructed and used to infect cell lines and primary human breast cancer cells. Assays for fusion and cell killing will identify the most efficient F protein variant for killing of breast cancer cells. In aim 2, we will construct plasmids encoding F protein variants engineered to contain cleavage sites for MMPs, cell surface proteases that have been shown to be overexpressed on a number of breast cancer cells. Biochemical and microscopic assays for fusion and cell killing will be carried out on transfected cells to test the hypothesis that these F proteins are cleavage activated in cells that overexpress these proteases. In the third aim, we will create conditionally-replicating rSV5 vectors whose growth and cell killing is restricted to tumor cells that overexpress MMPs. The ability to induce cell-cell fusion and cell death will be assayed using cultured cell lines that vary in levels of MMP proteinases. Mixing experiments will determine the extent to which rSV5-F-MMP vectors are restricted to cancer cells versus normal cells.
The work proposed here is aimed at addressing breast cancer cell targeting by engineering the SV5 F to be activated by proteases overexpressed in tumor cells. At the conclusion of these studies, we will have established the proof-of-principle needed to move our studies into mouse model systems and ultimately into clinical trials. These novel vectors will provide new approaches to treatment of breast cancer, with a preference for killing cells with properties of metastatic tumor cells.
Novel breast cancer therapeutic vectors based on a paramyxovirus fusogenic protein.
Viruses and virus-based vectors represent powerful tools for delivery of therapeutic molecules to specific cells or tissues. Within the past few years, the new technology for engineering RNA viruses has raised the exciting possibility that these viruses can be used for cancer therapy. However, a distinct and widely recognized limitation of current virus-based systems has been the lack of success in engineering viruses to kill only cancer cells or tumors with little or no effect on normal cells. This proposal seeks funding to fill this gap by developing a novel RNA virus system for selective killing of breast cancer cells.
A highly innovative aspect of the work proposed here is the focus on Simian Virus 5 (SV5) as novel therapeutic vector. SV5 is a member of a group of enveloped viruses containing a genome composed of RNA. Paramyxoviruses are common organisms involved in acute respiratory and systemic infections. This diverse group includes a number of human respiratory pathogens such as human parainfluenza viruses, respiratory syncytial virus (RSV), and measles virus. SV5 has great potential as an RNA virus vector, because the virus can infect humans and non-human primates, but is not associated with known disease. Importantly, SV5 infection of cells does not result in cell death. This property has allowed us to engineer SV5 to selectively kill breast cancer cells in a controlled way and by mechanisms that are relatively well understood. In this proposed work, we will engineer SV5 to have a strong preference for infection and killing of breast cancer cells, particularly those cancer cells that have properties of metastatic tumor cells.
Our idea is based on properties of the SV5 proteins that are needed for initiating infection of cells. SV5 entry into cells requires a viral protein termed the fusion or F protein. In addition to functioning in cell entry, F protein can also promote fusion of an infected cell expressing F protein with a neighboring uninfected cell. This cell-cell fusion creates multinucleated “giant cells” or syncytia. Ultimately, these syncytia die. Importantly, one infected cell can spread the F protein to neighboring cells to initiate spread of cell death. This type of cell killing has advantages for tumor therapy, since not only does the infected cell die, but dead cells induce a potent inflamatory response that could further aid in tumor clearance.
The SV5 F protein is unique among viral proteins, and we will exploit these unique properties to create novel therapeutics for breast cancer. The activity of SV5 F is controlled by cellular proteases that nick the F protein at a single site to activate the fusion process. A large number of studies have shown that breast cancer cells overexpress proteases, many of which are associated with highly invasive or metastatic tumors. Our goal is to engineer the SV5 F protein to be cleavage-activated by cellular proteases that are overexpressed in breast cancer cells relative to normal cells.
The overall objective of the proposed work is to develop the paramyxovirus SV5 as a therapeutic vector for controlled killing of breast cancer cells. We will test the hypothesis that our novel F proteins will fuse cells that overexpress tumor-associated proteases. Novel recombinant viruses harboring these new cleavage-activated F proteins will be developed to test the hypothesis that the specificity of SV5 infection has been changed to heavily bias growth in tumor cells and cell killing by F-mediated fusion.
There is great promise for the use of viral vectors for targeted therapy of breast cancer, and this project represents an innovative approach to achieve this goal using engineered SV5. At the conclusion of these studies, we will have established the proof-of-principle needed to move our studies into mouse model systems and ultimately into clinical trials.