Development of gene therapy for progressive renal diseases by ribozyme

为了开发针对进展性肾脏疾病的核酶治疗方法，我们利用DNA/RNA合成仪设计并合成了针对PDGF A链和转化生长因子-β1的核酸抗性DNA/RNA嵌合核酶。此外，我们还获得了针对PDGF A链和转化生长因子-β1腺病毒载体的重组核酶。用Zelatin递送试剂将FITC标记的DNA/RNA嵌合核酶大量导入培养的肾小球系膜细胞。在体外实验的基础上，我们研究了针对PDGF A链和转化生长因子-β1的DNA/RNA嵌合核酶的体内传递及其对Dahl盐敏感大鼠进展性肾脏疾病的影响。静脉注射FITC标记的DNA/RNA嵌合核酶后，可将其足量注入Dahl盐敏感大鼠的肾小球和肾小管。含Zelatin递送试剂的DNA/RNA嵌合核酶和重组核酶显著抑制肾皮质PDGF A链和转化生长因子-β1的mRNAs和蛋白的表达，并使尿蛋白排泄量减少一半。静脉注射过量的DNA/RNA嵌合核酶和Zelatin递送试剂及重组核酶未引起脾、骨髓、睾丸、肾脏和心脏的组织损伤，表明核酶治疗在体是安全的。这些结果表明，以Zelatin递送试剂为靶点的DNA/RNA嵌合核酶和针对PDGF A链和TGF-β1的重组核酶将成为治疗进展性肾脏疾病的可行的基因治疗方法。

To develop ribozyme therapy for progressive renal diseases, we designed and synthesized the nucleic acid resistant DNA/RNA chimeric ribozyme targeting PDGF A-chain and TGF-b1 by DNA/RNA synthesizer. In addition, we also obtained the recombinant ribozyme targeting PDGF A-chain and TGF-b1 adeno virus vector. FITC-labeled DNA/RNA chimeric ribozymes were considerably delivered into cultured mesangial cells with zelatin delivery reagent. The ribozymes significantly inhibited expression of PDGF A-chain and TGF-b1 mRNA in mesangial cells in vitro.Based on the in vitro experimental results, we investigated in vivo delivery and effects of the DNA/RNA chimeric ribozyme targeting PDGF A-chain and TGF-b1 on progressive renel diseases in Dahl-salt sensitive rats. FITC-labeled DNA/RNA chimeric ribozymes injected intravenously were sufficinatly delivered into gromeruli and nephrotubulus in Dahl-salt sensitive rats. The DNA/RNA chimeric ribozymes with the zelatin delivery reagent and the recombinant ribozymes significantly inhibited expressions of PDGF A-chain and TGF-b1 mRNAs and proteins in renal cortex, and decreased urinary protein excressions to half levels. Intra venous injections of excess amount of the DNA/RNA chimeric ribozymes with the zelatin delivery reagent and the recombinant ribozymes did not induce tissue damages in spleen, bone marrow, testis, kideney and heart, indicating that safety of the ribozyme treatments in vivo.These results suggest that the the DNA/RNA chimeric ribozymes with the zelatin delivery reagent and the recombinant ribozymes targeting PDGF A-chain and TGF-b1 will be feasible gene therapy for the progressive renal diseases.