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.