Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts
Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts
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DOI：
10.1093/nar/28.20.4013
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发表时间：
2000-10-15
影响因子：
14.9
通讯作者：
Marini, JC
中科院分区：
文献类型：
--
作者：
Dawson, PA;Marini, JC
核酶是一种很有前途的药物，通过等位基因特异性mRNA抑制显性阴性遗传病的基因治疗。为了测试细胞中的等位基因特异性mRNA抑制，我们使用来自成骨细胞增生症（OI）患者的成纤维细胞。这些细胞在一个α 1（I）胶原蛋白等位基因中含有突变，该突变既引起骨骼疾病又产生新的核酶切割位点。在初步的体外试验中，核酶切割突变体RNA底物，而正常底物保持完整。对于细胞培养中的研究，我们产生了稳定表达靶向突变型α 1（I）胶原mRNA的活性（AR）和非活性（IR）核酶的细胞系。I型胶原mRNA的定量竞争性RT-PCR分析（标准化为β-肌动蛋白表达水平）显示，在表达AR的细胞中，突变型α 1（I）胶原mRNA的水平显著降低约50%。当AR或IR从pH β APr-1-neo载体表达时，正常α 1（I）胶原mRNA显示无显著减少，而当任一核酶从pCl.neo载体表达时，正常α 1（I）胶原mRNA显示小的（10-20%）但显著的减少。在从表达AR的细胞衍生的克隆系中，核酶表达水平与突变体：正常α 1（I）mRNA比率的降低程度相关，范围为0.33至0.96。通过生长速率评估，活性核酶的稳定表达不影响细胞活力。核酶切割突变mRNA导致突变I型胶原蛋白减少，如SDS-尿素-PAGE所示。这是第一次报道核酶引起来自显性阴性遗传病患者的细胞中内源性突变mRNA的特异性抑制。
Ribozymes are a promising agent for the gene therapy of dominant negative genetic disorders by allele-specific mRNA suppression. To test allele-specific mRNA suppression in cells, we used fibroblasts from a patient with osteogenesis imperfecta (OI), These cells contain a mutation in one alpha1(I) collagen allele which both causes the skeletal disorder and generates a novel ribozyme cleavage site. In a preliminary in vitro assay, ribozymes cleaved mutant RNA substrate whereas normal substrate was left intact. For the studies in cell culture we generated cell lines stably expressing active (AR) and inactive (IR) ribozymes targeted to mutant alpha1(I) collagen mRNA, Quantitative competitive RT-PCR analyses of type I collagen mRNA, normalized to p-actin expression levels, revealed that the level of mutant alpha1(I) collagen mRNA was significantly decreased by similar to 50% in cells expressing AR. Normal alpha1(I) collagen mRNA showed no significant reduction when AR or IR was expressed from the pH beta APr-1-neo vector and a small (10-20%) but significant reduction when either ribozyme was expressed from the pCl.neo vector. In clonal lines derived from cells expressing AR the level of ribozyme expression correlated with the extent of reduction in the mutant:normal alpha1(I) mRNA ratio, ranging from 0.33 to 0.96, Stable expression of active ribozyme did not affect cell viability, as assessed by growth rates. Ribozyme cleavage of mutant mRNA results in a reduction in mutant type I collagen protein, as demonstrated by SDS-urea-PAGE, This is the first report of ribozymes causing specific suppression of an endogenous mutant mRNA in cells derived from a patient with a dominant negative genetic disorder.