Site-specific correction of hemoglobinopathies in iPS cells

Abstract

The development of somatic reprogramming has been studied for years. In 2006, the Yamanaka lab reported a new method to produce pluripotent stem cells from somatic cells with the help of exogenous expression of transcription factors. These new cells are called induced pluripotent stem cells (iPS). iPS cell technology holds vast promises to cure genetic diseases. We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. iPS cells produced from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells, show that regardless of the age of the donor cells, the iPSC produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hES cells based and low and high-throughput expression analysis and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with hematopoietic differentiation regardless of the age or the tissue type of the donor cells. The ability to produce large numbers of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications. Induced pluripotent stem cell technology holds vast promises for a cure to the hemoglobinopathies. Constructs and methods to safely insert therapeutic genes to correct the genetic defect need to be developed. Site-specific insertion is a very attractive method for gene therapy because the risks of insertional mutagenesis are eliminated provided that a "safe harbor" is identified, and because a single set of validated constructs can be used to correct a large variety of mutations simplifying eventual clinical use. We report here the correction of athalassemia major hydrops fetalis in transgene-free iPS cells using zinc finger-mediated insertion of a globin transgene in the AAVS1 site on human chromosome 19. Homozygous insertion of the best of the four constructs tested led to complete correction of globin chain imbalance in erythroid cells differentiated from the corrected iPS cells.