Development of gene therapy for progressive renal diseases by ribozyme

为了开发进展性肾脏疾病的核酶治疗方法，我们设计并合成了靶向PDGF a链和TGF-b1的核酸耐药DNA/RNA嵌合核酶。此外，我们还获得了靶向PDGF a链和TGF-b1腺病毒载体的重组核酶。用zelatin递送试剂将fitc标记的DNA/RNA嵌合核酶大量递送到培养的系膜细胞中。核酶可显著抑制系膜细胞PDGF a链和TGF-b1 mRNA的表达。基于体外实验结果，我们研究了靶向PDGF a链和TGF-b1的DNA/RNA嵌合核酶在达尔盐敏感大鼠进行性肾疾病中的体内递送和作用。经静脉注射fitc标记的DNA/RNA嵌合核酶，可在达尔盐敏感大鼠肾小球和肾小管中充分传递。DNA/RNA嵌合核酶与zelatin递送试剂和重组核酶可显著抑制肾皮质PDGF a链和TGF-b1 mrna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嵌合核酶与zelatin递送试剂和重组核酶在脾脏、骨髓、睾丸、肾脏和心脏均未引起组织损伤，表明该核酶治疗在体内是安全的。这些结果表明，结合zelatin递送试剂的DNA/RNA嵌合核酶和靶向PDGF a链和TGF-b1的重组核酶将是治疗进展性肾脏疾病的可行基因疗法。

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.