Development and characterisation of a zinc finger nuclease specific for the human beta-globin gene

Abstract

β-thalassemia and sickle cell disease, which are caused by mutations in the β-globin gene, are two of the most common single gene disorders worldwide and the only available cure is allogeneic bone marrow transplantation that is limited by donor availability. Gene therapy, by delivery of a β-globin expression vector into autologous haematopoietic stem cells, is a valuable alternative but the technique is affected by unpredictable protein expression levels and, more significantly, by random integration of the vector and the risk of insertional oncogenesis. Gene correction by Homologous Recombination (HR) with a DNA repair template would avoid the above-mentioned issues, becoming ideal therapeutic approach. Although spontaneous HR events are very rare, Double Strand Breaks (DSBs) at the target locus can greatly stimulate them. Therefore, the development of Zinc Finger Nucleases (ZFNs), which are customised endonucleases capable of cleaving any desired DNA sequence, has created the opportunity to design molecular tools to treat many rare monogenic disorders. The aim of this thesis has been to develop a ZFN-based gene targeting at the β-globin locus as a basis for β-thalassaemia gene-correction therapy. Initially, six ZF domains specific for the β-globin gene were designed, assembled and tested using publicly available reagents, but these were found to have a low binding efficiencies. Therefore, a commercial approach was used to obtain a functional ZFN (ZFN4) and this was shown to produce DSBs at the β-globin gene in ≈ 1% of the transfected human erythroleukemia cells without detectably cleaving the most closely related sequence in the human genome (the δ-globin gene). Using a reporter assay, ZFN4 was also shown to promote gene correction through intrachromosomal HR but was found to be 20 times less efficient than the homing endonuclease I-SceI. Ultimately, ZFN4-stimulated targeted integration of a drug resistance marker at the endogenous β -globin gene locus was tested: 95% of the drugresistant cells were targeted while the absolute frequency compared to the whole cell population resulted to be 0.1% The collected data show that ZFN4-mediated gene targeting of the β-globin locus is possible but further studies are required in order address the discrepancy between cutting and targeting efficiencies and to increase the absolute frequency of gene targeted cells.