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构建体改善骨生成障碍（OI）表型特征的能力。我们建议，在所有旨在纠正这些患者胶原缺陷的策略中，必须考虑与最严重形式的OI相关的“结构性”或非排除性I型胶原突变的显性负效应，并且消除突变等位基因必须非常有效，因为即使是低水平的突变等位基因产物也会产生主要的有害影响。本提案中描述的实验旨在推进我们开发的核酶基因，有效地靶向I型胶原突变，通常在严重的OI患者中观察到。特别地，将设计和测试自切割多聚体核酶表达基因。这些可以提供足够高的核酶递送到靶细胞，使得可以将更大程度的等位基因选择性设计到最终靶向杂合单核苷酸取代的核酶亚基中。在该提案中描述的实验包括：（1）设计和测试自切割多聚锤头状核酶，其选择性切割邻近人proal（I）胶原蛋白小基因pMG 155中的独特缺失连接。将通过体外RNase保护进行核酶效率的初步测试。靶构建体将在MC 3 T3-E1细胞中稳定表达，用于细胞内核酶测试。将通过北方和Western印迹分析、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表达小基因的MC 3 T3-E1细胞中明确定义的生化表型的切割效力和逆转。将表达pMG 155或最佳多聚体核酶表达基因的转基因小鼠交配，并将双转基因小鼠的表型与仅表达靶序列或核酶的小鼠的表型进行比较;（2）将对多聚体核酶进行类似的体外、细胞内和体内测试，其靶向在修饰形式pMG 155中产生的甘氨酸突变，所述修饰形式pMG 155含有来自鼠colla 2基因的三螺旋结构域的3'末端的符合读框的270 bp eDNA片段;和（3）我们将在胶原蛋白小基因和靶向小基因和骨钙蛋白的最佳核酶构建体的转基因小鼠中测试靶向OI显性阴性表型的潜在下游介体骨钙蛋白的可能的附加治疗益处。

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.