CHIMERIC RNA/DNA OLIGONUCLEOTIDE BASED GENE THERAPY

这项建议的长期目标是发展基因治疗遗传性肾脏疾病。尽管进行了大量的研究工作，但对于遗传性疾病，目前大多数基于使用病毒或非病毒载体表达正常全长cDNA的基因治疗方案在临床上并不有用。这主要是由于缺乏转基因的持续表达。利用嵌合RNA/DNA寡核苷酸的新方法已被提出在现场纠正突变基因。与传统的基于表达载体的基因治疗相比，这种基因靶向技术具有长期表达、细胞特异性、基因表达正常生理调控等巨大优势，避免了插入突变和病毒产物免疫反应等副作用。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，由这个和其他实验室。有了这些令人鼓舞的结果，研究人员建议在实验动物模型——碳酸酐酶II （CA-II）缺乏的小鼠中测试这种新策略。这些小鼠在CA II基因中有一个点突变，导致肾小管酸中毒。研究人员建议通过测试不同的途径和脂质体，通过修饰不同长度的寡核苷酸和核苷酸类似物，以及通过重复注射来开发一种最佳的基因靶向系统，以改善肾脏中的基因转化。将进行长期研究，以证明该方法提供持久的、功能性的、细胞特异性的和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抗体的形成和不相关基因高度同源序列中不必要的基因转化。新合成的CA II对插层细胞分化的影响以及对CA IV和磷酸钠共转运体-2的调控作用将被评价。这项临床前研究将为未来开发治疗遗传性肾脏疾病（如常染色体显性多囊肾病）的最佳基因靶向治疗提供重要信息。

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.