OLIGONUCLEOTIDE BASED GENE THERAPY FOR SICKLE CELL ANEMIA

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(Adapted from the Applicant's Abstract) Sickle cell anemia is the prototype of a genetic disease caused by a single base-pair mutation, an A-to-T transversion in the sixth codon of the human globin gene. At low oxygen tensions, the substitution of a single amino acid (GLU>Val) in the beta-globin subunit of hemoglobin results in a polymerization of HB S and leads to irregular shaped erythrocyte cells. The sickled erythrocyte cells become trapped n the microcirculation, causing extreme pain and damage to multiple organs. The investigators propose to test the hypothesis that triplex-forming oligonucleotides linked to mutagenic agents can be used to generate mutations in betaS and gamma-globin genes to inhibit the polymerization of HB S within erythrocyte cells and this may be utilized in gene therapy for sickle cell diseases. SV40-based shuttle vectors carrying the target genes will be constructed; an improved mammalian cell mutation assay system will be developed to facilitate the study of triplex-directed mutagenesis of the genes in vivo; oligonucleotides that bind to target sites will be designed and synthesized; oligonucleotide characteristics (such as nucleotide composition, chemical modifications, and analog substitutions) and targeted mutagenesis will be examined. Finally, experiments will be carried out to direct test the hypothesis that targeted mutagenesis of betaS and gamma-globin genes mediated by triplex-forming oligonucleotides can be achieved in vivo in the chromosomal DNA of mammalian cells in culture. The ultimate goal is the delivery of mutagenic oligonucleotides to bone marrow cells and the introduction of permanent and inheritable mutations into desired sites of the betaS and/or gamma-globin gene so that the polymerization of Hb S within cells will be inhibited. Although it may be some time before this technology is applied clinically, this grant application proposes a body of work to establish the potential for the role of targeted mutagenesis in both clinical and scientific endeavors.