CHIMERIC RNA/DNA OLIGONUCLEOTIDE BASED GENE THERAPY

点击翻译按钮获取中文摘要

The long-term goal of this proposal is to develop gene therapy for treating hereditary renal diseases. In spite of tremendous research effort, for hereditary diseases, most of current gene therapy protocols, which are based on the expression of a normal full-length cDNA using either viral or non-viral vectors, are not clinically useful. This is mainly due to the lack of persistent expression of transgenes. Novel approaches using chimeric RNA/DNA oligonucleotides have been proposed to correct the mutated genes on site. This gene targeting technique has tremendous advantages over the traditional expression vector- based gene therapy, including long term expression, cell specificity, and normal physiological regulation of gene expression and avoids side effects such as insertion mutagenesis and immune reactions to viral products. Gene targeting with chimeric RNA/DNA oligonucleotides has been achieved in cultured cells, and more recently in animals, by this and other laboratories. With these encouraging results, the investigators propose to test this new strategy in an experimental animal model, carbonic anhydrase II (CA-II) deficient mice. These mice have a point mutation in the CA II gene that results in renal tubular acidosis. The investigators propose to develop an optimal gene targeting system to improve gene conversion in the kidney by testing different routes and liposomes, by modifying oligonucleotides with different lengths and nucleotide analogs, and by repeated injections. Long-term studies will be performed to prove that this approach provides persistent, functional, cell specific, and normally regulated gene expression, and to assess potential side effects, including biochemistry and histological changes, formation of anti-CA II antibodies and unwanted gene conversion in highly homologous sequences of unrelated genes. The effects on newly synthesized CA II on differentiation of intercalated cells and on regulation of CA IV and sodium, phosphate cotransporter-2 will be evaluated. This preclinical study will provide critical information for future development of optimal gene targeting therapy for treating hereditary renal diseases, such as autosomal dominant polycystic kidney disease.