Sickle cell anemia is one of the most common inherited blood disorders, affecting approximately 100,000 newborn each year. It is caused by a structural aberration in the essential oxygen-carrying protein hemoglobin that leads to malformation of the red blood cells, resulting in painful episodes and early death. This malformation is the result of a single amino acid substitution (glutamic -> acid valine) at position 6 of the beta-globin chain, which, in turn, is due to an A ->T transversion mutation on chromosome 11. Correction of this mutation even at the somatic gene level can effectively prevent the disease, and partial correction is enough to dramatically ease the symptoms. The goal of this project is to develop a sickle cell gene repair approach that takes advantage of the specificity of nucleic acid third-strand binding to deliver a reactive moiety, e.g., UV photoreactive psoralen adjacent to the T residue of the mutant base pair. T ->A transversion at the Sickle Cell mutation site can then be triggered via recognition and excision of the psoralen adduct by the cellular DNA repair machinery. A high degree of such directed psoralen modification can ultimately result in replacement of the mutant base pair by a wild type one with sufficient efficiency to restore satisfactory levels of normal betaglobin synthesis. Specific third-strand binding and photoadduct formation, the first step towards the desired goal, i.e., has already been achieved in vitro using a 40 bp linear DNA model of the mutant sequence of the Sickle beta- globin gene. It is now planned to extend this binding to a plasmid containing the full beta-globin target sequence in order to achieve enzymatic repair of the photomodified plasmid both in vitro and in vivo in mammalian cell lines. This technology will then be extended to third- strand binding to chromosome 11 in human erythroleukemia cell lines that can differentiate into erythroid cells. Methods for third-strand delivery into other cell lines will also be investigated. This research will require assessment of normal hemoglobin expression and efficiency of gene correction. By these different steps, it is hoped to develop a complete gene repair system particular to the Sickle Cell hemoglobin target, that can eventually be applied to human hematopoietic stem cells ex vivo. This technology is potentially applicable to other inherited human hemoglobin- based anemias and a range of other point mutation-based diseases.
