NOVEL ANTI-SICKLING STRATEGIES: GLOBINS AND RIBOZYMES

该项目直接源于前一个赠款期间的成就：a)制定了提高抗镰刀珠蛋白有效性的策略；b)在转基因小鼠中证明了核酶策略减少HBS合成的潜力。在这项涉及新研究人员(Dr.Bouhassira and Jian-Yang Wang)的提案中，我们首先致力于全面开发和鉴定第二代抗镰状珠蛋白，其中包括通过结合使用α和β抗镰状突变构建比HBF更具抑制性的超级抗镰状珠蛋白，以及利用比天然HBF具有更高抑制潜力的动物珠蛋白链。第二种策略是赋予抗镰状抗镰状珠蛋白有利于α1β1二聚体的形成，从而通过有利于它们的组装来改善它们的表达。第三种策略是创造具有低O2亲和力的抗病珠蛋白，以确保它们优先脱氧，使未经修饰的HBS四聚体充氧和无害。本项目所需的新转基因系的产生和抗镰刀菌特性的验证将与Core B和Project 2合作进行。第二个主要的新目标是通过优化抗Beta5-珠蛋白锤头状核酶活性来进一步开发核酶方法，这一事实支持了以下事实：由于HBS聚合反应的生物物理学，只需少量减少Beta5-珠蛋白就能产生显著的临床效果。我们将努力通过计算分析和在体外和细胞研究中测试这些结果的组合来确定纯化的HBS mRNA中最容易受到核酶攻击的位置。我们还将确定哪些已确定的位置适合细胞内攻击，并利用这些知识构建核酶表达盒，随后在镰刀鼠模型中测试核酶表达盒。其次，世代抗病和核酶策略并不是相互排斥的，事实上，最成功的策略可以组合成一个多时间延长的抗病磁带。这些策略最终将在小鼠和人类细胞的转基因实验以及与项目3和4协调的移植实验中进行测试。

This project stems directly from the accomplishments of the previous grant period: a) The development of strategies to increase the effectiveness of anti-sickling globins; b) Proof of principle in transgenic mice of the potential of a ribozyme strategy to decrease the synthesis of HbS. In this proposal, which involves new investigators (Drs. Drlica, Bouhassira and Jian-Yang Wang) we aim first at fully developing and characterizing second generation anti-sickling globins, this includes the construction of super-anti-sickling globins that are more inhibitory than HbF by using a combination of alpha and beta anti-sickling mutations and taking advantage of animal globin chains that have higher inhibitory potential than native HbF. A second strategy is endowing anti-sickling anti-sickling globins with advantage towards alpha1beta1 dimer formation hence improving their expression by favoring their assembly. A third strategy is the creation of anti-sickling globins with low O2 affinity to ensure their preferential deoxygenation, leaving the un-modified HbS tetramers oxygenated and harmless. The generation of new transgenic lines required in this project and the validation of anti-sickling properties will occur in collaboration with Core B and Project 2. A major second and novel aim is to further develop the ribozyme approach by optimizing the anti-beta5- globin hammerhead ribozyme activity, supported by the fact that only a small reduction in beta5-globin is needed to have a significant clinical effect due to the biophysics of the HbS polymerization reaction. We will strive to identify the most accessible sites in purified HbS mRNA for ribozyme attack by a mixture of computational analysis and testing these results in vitro and in cell studies. We will also determine which sites identified are suitable for intracellular attack and with this knowledge construct and subsequently test ribozyme expression cassettes in sickle mouse model. Second, generation anti-sickling and ribozyme strategies are not mutually exclusive and indeed the most successful ones can be combined into a multi-prolonged anti-sickling cassette. The strategies will eventually be tested in transfection experiment of murine and human cells and in transplantation experiments in coordination with Project 3 and 4.