Our ultimate goal is to develop targetable, injectable vectors for the delivery of therapeutic genes to specific cell populations for application to the treatment of cancer and other diseases. Targetable vectors based upon murine leukemia virus (MLV) vectors should have the ability to bind to restricted cell populations, while retaining the ability of MLV to catalyze the fusion of viral and cell membranes. We will build chimeric envelope proteins by replacing the receptor-binding sequences of MLV with segments of other proteins that are capable of binding to specific cell- surface proteins. The goal of this project is to understand the mechanisms by which MLV vectors bind and fuse with cells. We believe that increased knowledge of the relationship between binding and fusion will enable us to build chimeric envelope proteins that will allow retroviral vector particles to transduce cells at least as efficiently as the MLV vectors currently in use. Quantitative assays to measure the affinity of binding and its kinetics, and to measure the efficiency of fusion will be developed. They will be used to evaluate the consequences of making a series of site-directed changes in the binding domains of the ecotropic and amphotropic envelope proteins. These quantitative data should permit us to generate a map of the amino acids within the envelope that play direct roles in receptor binding and in catalyzing the process of membrane fusion. In doing so we should gain insight into how the binding of MLV to its receptor triggers membrane fusions. In doing so we should gain insight into how the binding of MLV to its receptor triggers membrane fusion. We plan to produce soluble monomeric receptor binding domains from the ecotropic and amphotropic envelope proteins, and use them to characterize the number and homogeneity of receptors for these viruses on a variety of cell types. We will provide the soluble amphotyropic and ecotropic envelope binding-domain proteins to our collaborator (David Eisenberg, UCLA) who will use them to try to determine their three-dimensional structures by X-ray diffraction.
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