CHIMERIC RNA/DNA OLIGONUCLEOTIDE BASED GENE THERAPY

这项提议的长期目标是开发治疗遗传性肾脏疾病的基因疗法。尽管有大量的研究工作，但对于遗传性疾病，目前的大多数基因治疗方案都是基于病毒或非病毒载体表达的正常全长cDNA，并不能在临床上使用。这主要是由于转基因缺乏持续表达所致。已经提出了利用嵌合RNA/DNA寡核苷酸来现场纠正突变基因的新方法。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，并避免了插入突变和对病毒产物的免疫反应等副作用。这个实验室和其他实验室已经在培养细胞中实现了利用嵌合RNA/DNA寡核苷酸的基因靶向，最近在动物中也实现了这一点。有了这些令人鼓舞的结果，研究人员建议在实验动物模型--碳酸氢酶II(CA-II)缺陷小鼠身上测试这一新策略。这些小鼠的CA II基因点突变会导致肾小管性酸中毒。研究人员建议开发一种最佳的基因打靶系统，通过测试不同的路线和脂质体，通过修改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提高肾脏的基因转化。将进行长期研究，以证明该方法提供持续的、功能的、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，包括生化和组织学变化、抗CA II抗体的形成以及高度同源的无关基因序列中不必要的基因转化。将评估新合成的CA II对细胞分化的影响，以及对CA IV和钠、磷协同转运体2的调节作用。这项临床前研究将为未来开发治疗遗传性肾脏疾病的最佳基因靶向疗法提供关键信息，例如常染色体显性遗传性多囊肾病。

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.