NOVEL ANTI-SICKLING STRATEGIES: GLOBINS AND RIBOZYMES

这个项目直接源于上一个赠款期取得的成就：a)制定提高抗镰状球蛋白有效性的战略；b)在转基因小鼠中证明核酶策略可能减少HbS合成的原理。在这个提议中，涉及到新的研究者(dr。Drlica， Bouhassira和汪建阳)，我们的目标首先是充分开发和表征第二代抗镰状球蛋白，这包括利用α和β抗镰状球蛋白突变的组合，利用比天然HbF具有更高抑制潜力的动物珠蛋白链，构建比HbF更具抑制作用的超抗镰状球蛋白。第二种策略是赋予抗镰状抗镰状球蛋白有利于形成α - β - 1二聚体，从而通过有利于其组装来改善其表达。第三种策略是创造具有低氧亲和力的抗镰状球蛋白，以确保其优先脱氧，使未修饰的HbS四聚体氧化且无害。本项目所需的新转基因品系的产生和抗镰状病特性的验证将与Core B和project 2合作进行。第二个主要的新目标是通过优化抗β 5-珠蛋白锤头核酶活性来进一步发展核酶方法，因为由于HbS聚合反应的生物物理特性，只需少量减少β 5-珠蛋白就可以产生显著的临床效果。我们将通过计算分析和在体外和细胞研究中测试这些结果，努力确定纯化HbS mRNA中最易接近的核酶攻击位点。我们还将确定哪些位点适合细胞内攻击，并利用这些知识构建并随后在镰状小鼠模型中测试核酶表达盒。第二，一代抗镰状细胞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延长的抗镰状细胞盒。这些策略最终将在小鼠和人类细胞的转染实验以及与项目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.