FUNCTIONAL RIBOZYME EXPRESSION IN VIVO

细胞内生产核酶以特异性破坏HIV RNA靶标代表了一种潜在的令人兴奋的基因治疗形式 抗感染或激活潜伏病毒。催化剂 几种小核酶的性质正在受到严格的审查 体外试验，但有大量的参数需要 如果合理的设计标准要在体内进行系统研究 公式化的。这些包括高效表达、稳定性、 正确的折叠，细胞定位和微小的变化 在生理条件下的退火和催化性能。 在这项建议中，最佳核酶表达盒的模型 将利用酵母中的遗传选择的力量来开发 酿酒酵母。在酵母菌中，可以选择 通过靶向核酶发挥高效的核酶功能 几个mRNAs和针对目标基因产物的选择。二 将使用不同的RNA聚合酶III启动子来表达 “锤头”和“发夹”核酶变异体的微型文库 针对来自三个这样的mRNA中的多个靶点 基因、LYS2、URA3和CAN1。有效地销毁任何一个低 或高拷贝靶基因经文库转化后即可 被选择性媒体的增长所要求。的属性 在体内产生高水平真实核酶活性的构建 VIVO将被确定并用于制定预测模型 合适的表达盒和核酶结构。这些 通用模型将作为构建表达式的基础 用于对抗HIV-1靶标的盒式磁带和核酶插入物。

Intracellular production of ribozymes to specifically destroy HIV RNA targets represents a potentially exciting form of gene therapy against infection or activation of latent virus. The catalytic properties of several small ribozymes are under intense scrutiny in vitro, but there are a large number of parameters that will require systematic study in vivo if rational design criteria are to be formulated. These include efficient expression, stabilization, correct folding, cellular localization, and minor changes in annealing and catalytic properties under physiological conditions. In this proposal models for optimum ribozyme expression cassettes will be developed using the power of genetic selections in the yeast Saccharomyces cerevisiae. It is possible in yeast to select for efficient ribozyme function by targeting the ribozymes against one of several mRNAs and selecting against the target gene product. Two distinct RNA polymerase III promoters will be used to express mini-libraries of "hammerhead" and "hairpin" ribozyme variants directed against multiple target sites in mRNAs from three such genes, LYS2, URA3, and CAN1. Efficient destruction of either low or high copy target mRNA after transformation with the libraries can be demanded by growth on selective media. The properties of constructs producing high levels of bonafide ribozyme activity in vivo will be determined and used to formulate predictive models for appropriate expression cassettes and ribozyme structures. These general models will serve as a basis for constructing expression cassettes and ribozyme inserts for use against HIV-1 targets.