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

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

A new cellular model to follow Friedreich's ataxia development in a time-resolved way

Friedreich's ataxia (FRDA) is a recessive autosomal ataxia caused by reduced levels of frataxin (FXN), an essential mitochondrial protein that is highly conserved from bacteria to primates. The exact role of frataxin and its primary function remain unclear although this information would be very valuable to design a therapeutic approach for FRDA. A main difficulty encountered so far has been that of establishing a clear temporal relationship between the different observations that could allow a distinction between causes and secondary effects, and provide a clear link between aging and disease development. To approach this problem, we developed a cellular model in which we can switch off/on in a time-controlled way the frataxin gene partially mimicking what happens in the disease. We exploited the TALEN and CRISPR methodologies to engineer a cell line where the presence of an exogenous, inducible FXN gene rescues the cells from the knockout of the two endogenous FXN genes. This system allows the possibility of testing the progression of disease and is a valuable tool for following the phenotype with different newly acquired markers

Label-free, real-time measurement of metabolism of adherent and suspended single cells by in-cell fourier transform infrared microspectroscopy

We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles

Analyzing the Effects of a G137V Mutation in the FXN Gene

Reduced levels of frataxin, an essential mitochondrial protein involved in the regulation of iron-sulfur cluster biogenesis, are responsible for the recessive neurodegenerative Friedreich Ataxia (FRDA). Expansion of a GAA triplet in the first intron of the FRDA is essential for disease development which causes partial silencing of frataxin. In the vast majority of cases, patients are homozygotes for the expansion, but a small number of FRDA patients are heterozygotes for expansion and point mutations in the frataxin coding frame. In this study, we analyze the effects of a point mutation G137V. The patient P94-2, with a history of alcohol and drug abuse, showed a FRDA onset at the border between the classic and late onset phenotype. We applied a combination of biophysical and biochemical methods to characterize its effects on the structure, folding and activity of frataxin. Our study reveals no impairment of the structure or activity of the protein but a reduced folding stability. We suggest that the mutation causes misfolding of the native chain with consequent reduction of the protein concentration in the patient and discuss the possible mechanism of disease

Adding a temporal dimension to the study of Friedreich's ataxia:the effect of frataxin overexpression in a human cell model

The neurodegenerative disease Friedreich's ataxia is caused by lower than normal levels of frataxin, an important protein involved in iron–sulfur (Fe-S) cluster biogenesis. An important step in designing strategies to treat this disease is to understand whether increasing the frataxin levels by gene therapy would simply be beneficial or detrimental, because previous studies, mostly based on animal models, have reported conflicting results. Here, we have exploited an inducible model, which we developed using the CRISPR/Cas9 methodology, to study the effects of frataxin overexpression in human cells and monitor how the system recovers after overexpression. Using new tools, which range from high-throughput microscopy to in cell infrared, we prove that overexpression of the frataxin gene affects the cellular metabolism. It also leads to a significant increase of oxidative stress and labile iron pool levels. These cellular alterations are similar to those observed when the gene is partly silenced, as occurs in Friedreich's ataxia patients. Our data suggest that the levels of frataxin must be tightly regulated and fine-tuned, with any imbalance leading to oxidative stress and toxicity

The role of interruptions in polyQ in the pathology of SCA1

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission