CHIMERIC RNA/DNA OLIGONUCLEOTIDE BASED GENE THERAPY

这项建议的长远目标是发展基因疗法治疗遗传性肾病。 尽管进行了大量的研究工作，但对于遗传性疾病，大多数目前的基因治疗方案（其基于使用病毒或非病毒载体表达正常全长cDNA）在临床上是无用的。 这主要是由于缺乏转基因的持续表达。 已经提出了使用嵌合RNA/DNA寡核苷酸的新方法来现场校正突变的基因。 这种基因打靶技术与传统的基于表达载体的基因治疗相比具有巨大的优势，包括长期表达、细胞特异性和基因表达的正常生理调节，并且避免了副作用如插入诱变和对病毒产物的免疫反应。 嵌合RNA/DNA寡核苷酸的基因靶向已经在培养细胞中实现，最近在动物中，通过这个实验室和其他实验室。 有了这些令人鼓舞的结果，研究人员建议在实验动物模型中测试这种新策略，即碳酸酐酶II（CA-II）缺陷小鼠。 这些小鼠在CA II基因中具有导致肾小管酸中毒的点突变。 研究人员建议开发一种最佳的基因靶向系统，通过测试不同的途径和脂质体，通过修饰具有不同长度和核苷酸类似物的寡核苷酸，以及通过重复注射来改善肾脏中的基因转换。 将进行长期研究，以证明这种方法提供了持久的，功能性的，细胞特异性的，正常调节的基因表达，并评估潜在的副作用，包括生物化学和组织学变化，抗CA II抗体的形成和不必要的基因转换在高度同源序列的无关基因。将评价新合成的CA II对嵌入细胞分化以及对CA IV和钠磷酸盐协同转运蛋白-2调节的影响。 这项临床前研究将为未来开发治疗遗传性肾病（如常染色体显性遗传性多囊肾病）的最佳基因靶向治疗提供关键信息。

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.