Annarita MICCIO

The ‘Chromatin and Gene Regulation during Development' laboratory focuses on two main areas: 1. The dynamics of transcriptional and epigenetic networks during stem cell development
Identifying regulatory regions that control gene expression programmes is fundamental to understanding the molecular mechanisms underlying many diseases and to developing new therapeutic approaches. Indeed, many disease-associated sequence variations occur at the level of cis-regulators, which in some cases represent potential therapeutic targets. The aim of our research is the pan-genomic analysis of regulatory sequences utilised by human haematopoietic and epidermal stem/progenitor cells and induced cell lines at different stages of development (Cavazza A. et al., Stem Cell Reports, 2016; Romano O. et al., Scientific Reports, 2016). The genetic and epigenetic profiles were characterised using a number of genomic and bioinformatic tools, including RNA-seq, deepCAGE, retroviral scanning and ChIP-Seq. This research has led to a better understanding of the molecular basis of ‘stemness' and the involvement of clinically relevant stem cell lineages, providing a knowledge base for the safer and more effective use of stem cells in cell and gene therapy (Romano O. et al., J Vis Exp. 2017; Romano O. et al., Stem Cells Translational Medicine, 2017).
We are currently analysing, on a pan-genomic scale, the role of haematopoietic transcription factors (e.g. GATA1 and GATA2) and their co-factors, as well as epigenetic modifications of histones associated with transcription or ‘silencing', in order to identify regulatory regions involved in haematopoietic stem cell biology and in erythroid commitment and differentiation. The validation of putative regulatory regions is carried out using CRISPR-Cas9 and chromatic conformation capture approaches (Romano et al., under review; Meneghini et al., in preparation). 2. Molecular approaches to the treatment of β-haemoglobinopathies
Sickle cell disease and β-thalassaemia are genetic disorders caused by mutations in the gene encoding the β-chain of haemoglobin in adults. They are the most common monogenic disorders worldwide and affect thousands of newborns every year. In β-thalassaemia, reduced production of β-chains leads to the precipitation of α-globin and the death of red blood cells. In sickle cell anaemia, a single amino acid substitution in the β-globin chain causes haemoglobin to polymerise and the red blood cells to become deformed, taking on a sickle-like shape.
β-haemoglobinopathies can lead to a severe clinical phenotype characterised by anaemia, pain crises and organ damage. To date, the only curative treatment is a bone marrow transplant from a compatible donor: this is only an option for fewer than 30 per cent of patients. 

Experimental treatments include gene therapy and pharmacological treatments. In particular, studies are currently underway to identify compounds that might increase the expression of foetal globin genes. The rationale behind this approach is based on the long-standing observation that patients carrying mutations that trigger high levels of foetal globin expression experience a more benign clinical course of the disease (Cavazzana, Antoniani and Miccio, Molecular Therapy, 2017). However, pharmacological treatments are not equally effective in all patients, are associated with significant toxicity and do not provide a definitive cure. Several nuclear factors, such as the master erythroid regulator GATA1, its co-factors FOG1 and BCL11A, and the NuRD repressor complex, are involved in inhibiting g-globin expression. However, their role in erythroid development and haemoglobin expression has not yet been fully elucidated. 

The aim of our research is to advance scientific understanding in order to develop safe therapies for sickle cell disease and β-thalassaemias, using approaches involving lentiviruses and genome editing to increase g-globin expression. We aim to characterise the transcription factors and regulatory genomic elements that control the transition from foetal to adult globin gene expression. Precise mapping of the regulatory elements involved in this transition may provide potential therapeutic targets for inducing foetal haemoglobin.

Our research focuses on the molecular mechanisms that enable the switch from β-globin to γ-globin, as well as on evaluating the efficacy and safety of therapeutic approaches targeting these mechanisms. We utilise both established and novel molecular techniques (e.g. pan-genomic analyses, lentiviruses and CRISPR/Cas9) with various cellular models, including clinically relevant haematopoietic stem cells (Antoniani, Meneghini et al., Blood, 2018; Lagresle-Peyrou C. et al., Haematologica, 2018; Lattanzi et al., Molecular Therapy, 2018; Weber et al., Molecular Therapy and Clinical Development, 2018; Weber, Frati et al., Science Advances, in press; Meneghini Ramadier et al., in preparation).