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裂多聚核酶结构改善成骨不全症（OI）表型特征的能力。我们建议，在纠正这些患者的胶原缺陷的所有策略中，必须考虑到与最严重的OI形式相关的“结构性”或非排除型I型胶原突变的显性负作用，并且必须高效地消除突变等位基因，因为即使低水平的突变等位基因产物也可能产生重大的有害影响。本提案中描述的实验旨在促进我们对核酶基因的开发，这些基因可有效靶向严重成骨不全患者中典型的I型胶原突变。特别是，自我切割多聚核酶表达基因将被设计和测试。这些可能为靶细胞提供足够高的核酶递送，因此可以设计更大程度的等位基因选择性核酶亚基，最终针对杂合单核苷酸取代。本提案中描述的实验包括：(1)设计和测试自切割多聚锤头核酶(s)，该酶选择性地切割人类proa1(I)胶原蛋白迷你基因pMG155中邻近的独特缺失连接。核糖酶效率的初步测试将在体外通过RNase保护进行。目标构建体将在MC3T3-E1细胞中稳定表达，用于细胞核酶检测。在表达minigene的MC3T3-E1细胞中，切割效果和明确的生化表型的逆转将通过Northern和Western blot分析、前胶原的脉冲追踪标记和几种细胞分化标志物的测量来证明。将表达pMG155或最优多聚核酶表达基因的转基因小鼠进行配对，并将双转基因小鼠的表型与仅表达目标序列或核酶的小鼠进行比较；(2)类似的体外、细胞内和体内测试将对多聚核酶进行，该多聚核酶针对的是在pMG155的修饰版本中产生的甘氨酸突变，pMG155含有来自小鼠colla2基因三螺旋结构域3'端的帧内270 bp eDNA片段；(3)我们将测试针对成骨不全显性阴性表型的潜在下游介质骨钙素的可能的附加治疗效益，在小鼠中转基因胶原蛋白和针对骨钙素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.