One of the most promising approaches to molecular therapy for cancer and viral infection is the use of antisense DNA. Antisense oligonucleotides are potent inhibitors of gene expression in vitro, and have been shown to block a number of cellular genes associated with tumor growth or viral infection in cultured cells. The chemical modification of double stranded oligonucleotides so that they can be administered systemically has now provided the means for molecular therapy in vivo. Unfortunately, the mechanisms responsible for antisense uptake in specific tissues remains unclear, although an area of intense investigation. When administered systemically, antisense oligonucleotides are taken up predominantly by liver and kidney. As a result, these two organs are ideal therapeutic targets but they will likely be the major sites of toxicity as well. To direct therapy both to and away from the kidney in the future, the molecular basis of antisense uptake must be defined. This proposal intends to address this critical issue by defining the molecular basis of antisense uptake in the kidney. We have already purified the major DNA binding protein in renal brush border membrane, we have established that this protein binds oligonucleotides in electrophoretic mobility shift assays, and we have demonstrated that this protein allows the conductance of current through an artificial lipid bilayer in the presence of oligonucleotides. Partial amino acid sequence has been determined and reveals the protein to be novel with partial homology to known transcription factors, DNA binding proteins, and transmembrane shuttle proteins.
The specific aims of this proposal are, therefore, straightforward: 1. To clone the full-length cDNA for the major oligonucleotide channel protein by using degenerate primers and guessmers deduced from known amino acid sequence; a renal epithelium cDNA library will be screened for this purpose. 2. To determine the functional characteristics of DNA channel activity in cell culture and in artificial lipid bilayers and to map the functional peptide domains through site- directed mutagenesis. and 3. To use the information obtained to design effective strategies to inhibit gene expression in vivo using an animal model of a single gene-induced form of renal failure. These studies should provide the needed experimental basis for proposing studies of systemic antisense treatment for renal diseases in man.
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