Use of Hammerhead Ribozymes in Murine Models of Ol

描述(由申请人提供)：这些研究的总体目标是测试自切割多聚体核酶结构改善成骨不全(OI)表型特征的能力。我们建议，在所有旨在纠正这些患者的胶原缺陷的策略中，必须考虑与最严重的OI形式相关的“结构性”或非排斥型I型胶原突变的显性-负面影响，并且消除突变等位基因必须是高效的，因为即使是低水平的突变等位基因产物也可能产生重大的有害影响。这项建议中描述的实验旨在促进我们开发核酶基因，有效地靶向在严重OI患者中观察到的I型胶原突变。特别是，将设计和测试表达自我切割的多聚体核酶基因。它们可以向靶细胞提供足够高的核酶递送，从而可以将更大程度的等位基因选择性设计成最终针对杂合单核苷酸替换的核酶亚基。这项建议中描述的实验包括：(1)设计和测试自切割多锤头状核酶(S)，它选择性地切割人ProA1(I)胶原蛋白微小基因pMG155中唯一的缺失连接点附近。核酶效率的初步测试将在体外通过核糖核酸酶保护进行。目的构建物在MC3T3-E1细胞中稳定表达，用于纤维素型核酶检测。通过Northern和Western印迹分析、原胶原蛋白的脉冲追逐标记以及几个细胞分化标志物的测量，将证明表达微型基因的MC3T3-E1细胞中明确定义的生化表型的切割效率和逆转。表达pMG155或最优多聚体核酶表达基因的转基因小鼠将进行交配，并且双转基因小鼠的表型将与仅表达靶序列或核酶的小鼠的表型进行比较；(2)类似的体外、细胞和体内测试将对多聚体核酶进行，该多聚体核酶针对在修饰版本pMG155中产生的甘氨酸突变，该多聚体核酶含有来自小鼠COLALA2基因三螺旋结构域3‘端的框内、270个碱基对的EDNA片段；以及(3)我们将测试靶向OI显性阴性表型的潜在下游介导物骨钙素在转基因小鼠中的可能的相加治疗益处，该转基因小鼠可获得微胶原基因和靶向微基因和骨钙素的最佳核酶构建。

DESCRIPTION (provided by applicant): The overall goal of these studies is to test the ability of self-cleaving multimeric ribozyme constructs to ameliorate phenotypic features of osteogenesis imperfecta (OI). We propose that the dominant-negative effect of "structural" or non-excluded type I collagen mutations associated with the most severe forms of OI must be taken into account in all strategies aimed at correcting the collagen defects in those patients, and that elimination of mutant alleles must be highly efficient, since even low levels of mutant allelic products can have a major deleterious effect. Experiments described in this proposal are designed to advance our development of ribozyme genes that effectively target type I collagen mutations typically observed in severe OI patients. In particular, self-cleaving multimeric ribozyme expressing genes will be designed and tested. These may provide sufficiently high ribozyme delivery to target cells so that a greater degree of allelic selectivity can be designed into ribozyme subunits that would ultimately be targeted against heterozygous single nucleotide substitutions. Experiments described in this proposal include: (1) design and testing of self-cleaving multimeric hammerhead ribozyme(s) that selectively cleave adjacent to a unique deletion junction in the human proa1(I) collagen minigene pMG155. Preliminary testing of the ribozymes' efficiency will be performed by RNase protection in vitro. Target constructs will be stably expressed in MC3T3-E1 cells for in cellulo ribozyme testing. Cleavage efficacy and reversal of a well-defined biochemical phenotype in minigene-expressing MC3T3-E1 cells will be demonstrated by Northern and Western blot analysis, pulse-chase labeling of procollagens, and measurement of several markers of cellular differentiation. Transgenic mice that express either pMG155 or the most optimal multimeric ribozyme expression gene will be mated, and the phenotypes of doubly transgenic mice will be compared to those of mice expressing only the target sequence or the ribozyme; (2) similar in vitro, in cellulo, and in vivo testing will be performed on multimeric ribozymes, that target glycine mutations created in modified versions pMG155 containing an in-frame, 270 bp eDNA segment from the 3' end of the triple helical domain of the murine colla2 gene; and (3) we will test possible additive therapeutic benefits of targeting a potential downstream mediator of the OI dominant negative phenotype, osteocalcin, in mice transgenic for a collagen minigene and optimal ribozyme construct targeting both the minigene and osteocalcin.