Viral delivery of modifiedsnRNAs corrects aberrant splicing of beta-globin pre-mRNA

In certain forms of β-thalassemia, mutations at positions 654, 705, and 745 of the second intron of the β-globin gene create new aberrant 5′ splice sites and activate the same 3′ cryptic splice site. The resulting inclusion of part of intron 2 into aberrantly spliced β-globin mRNA leads to translation of the truncated and non-functional form of β-globin protein. Work in this laboratory has shown that modified snRNAs containing sequences antisense to the 3 ′ cryptic splice sites in the β-thalassemic pre-mRNA reduced the incorrect splicing of pre-mRNA and led to increased levels of the correctly spliced mRNA and β-globin protein. In an effort to optimize this system, U7 snRNAs were modified to contain a sequence antisense to position 623, a region in between the 5′ and 3′ aberrant splice sites. Transfection of HeLa cells expressing the three thalassemic mutants with these modified U7 snRNAs targeted against position 623 led to higher levels of correction than those targeted against the 3′ cryptic splice site. In addition, U6 snRNAs were modified in a similar manner, restoring correct splicing of pre-mRNA and leading to increased levels of the correctly spliced β-globin mRNA.; Since the modified U6 and U7 snRNAs do not remove the mutation from the gene but instead repair the defective pre-nmRNA, they would require periodic administration throughout the lifetime of the thalassemic patient. To achieve permanent correction, the modified U6 and U7 snRNA genes were incorporated into lentiviral vectors. Transduction with the U7, but not the U6 lentiviral vector was able to restore correct splicing and translation of β-globin pre-mRNA in HeLa cell lines stably expressing the IVS2-654, IVS2-705 and IVS2-745 β-globin genes. Importantly, the therapeutic value of this system was demonstrated in hematopoietic stem cells and/or erythroid progenitor cells from a patient with IVS2-745/IVS2-1 thalassemia. Twelve days after transduction of the patient cells with the U7.623 lentiviral vector, the levels of correctly spliced β-globin mRNA and hemoglobin A were approximately 25 fold over background. These results should be regarded as a proof of principle for lentiviral vector based gene therapy for β-thalassemia.