Antisense Treatment for Thalassemia. Preclinical study.

DESCRIPTION (provided by applicant): The ultimate goal of this project is treatment of beta-thalassemia, a genetic blood disease, with antisense oligonucleotides. The project is based on the technology developed by the PI in which antisense oligonucleotides are used to restore correct functioning of a defective gene rather than, as commonly practiced, to inhibit the expression of an undesirable gene. Specifically, oligonucleotides are used to reverse aberrant splicing of human beta-globin pre-mRNA by blocking splice sites activated by a mutation IVS2-654 in intron 2 of the beta-globin gene. Aberrant splicing is also reversed for betaE mutation in exon 1. The oligonucleotides not only inhibit aberrant splicing but, by forcing the spliceosomes to form on the existing correct splice sites, restore correct splicing of beta-globin pre-mRNA, which is translated into the beta-globin polypeptide, ultimately restoring expression of hemoglobin. The main goal of this Phase I proposal is to improve the effectiveness of morpholino oligonucleotides targted to the aberrant IVS2-654 splice site by conjugating them with penetrating tat-like peptides. The proposed work will be carried mostly in erythroid cells from the IVS2-654 mice, in vivo in this mouse model of thalassemia and in cells from thalassemia patients with IVS2-654 thalassemia. The Specific Aims of the project are: 1) To test if morpholino-peptide conjugates effectively correct splicing of beta-globin pre-mRNA and restore expression of hemoglobin in vitro in erythroid cells from IVS2-654 mouse. 2) To test if morpholino-peptide conjugates effectively correct splicing of beta-globin pre-mRNA and restore expression of hemoglobin in vivo in IVS2-654 mouse. 3) To confirm that the most effective oligonucleotides identified in Specific Aims 1 and 2 are effective in erythroid cells from patients with thalassemia.