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.