Studio del meccanismo mediante cui la CsA interferisce con fattori di restrizione lentivirali indotti da interferone nelle cellule ematopoietiche umane

For efficacious gene transfer it is necessary to overcome different cellular barriers in order to introduce the new genetic information without compromising the essential biological functions of the treated cells. The corrected cells need to be present in enough quantity to replace the defective ones, capable to survive for a long term, to self-renew transmitting the modification to the progeny and nevertheless to escape immune recognition. Hematopoietic and progenitor stem cells (HSPCs) meet almost all of these properties and have been preferred target for ex-vivo LV-based gene therapy of several inherited and acquired diseases. HSPCs ex-vivo LV–based gene therapy consists of the transduction of the target cells, which has been isolated and purified from recipient patient, with a vector carrying an expression cassette for the corrective transgene.Recent clinical trials of gene therapy have shown remarkable therapeutic benefits and excellent safety records, however human hematopoietic stem and progenitor cells tend to be poorly transduced, and high vector doses, prolonged ex-vivo culture conditions and cytokines stimulation are required to reach clinically significant transduction levels. The reason of their resistance to transduction probably resides behind the fact that, although lentiviral vectors has been extensively modified and engineered to meet the safety requirements, they still share many common features with infectious HIV-1 virus from which they derive, most of all they rely on the same molecular mechanisms to reach the nuclear compartment of the target cells and integrate within the host genome. Several studies demonstrated that mammalian cells have evolved a mechanism of self-protection in order to block specific steps of retroviral life cycle, in particular host-antiviral factors referred to as restriction factors (RFs) have been discovered in cells of lymphoid and myeloid origin. RFs can be either ubiquitously expressed or induced by type I Interferon signaling. So far LVs can be recognized by innate sensors present in HSPCs which can activate specific pathways impacting transduction efficiency and/or the biological function of the host cell. In this context, many questions remain to be answered regarding the role and functions of innate host factors as well as the consequences of immune triggers on HSC biology and cell permissivity to gene transfer strategies involving LV. Therefore, efforts to improve LV transduction efficiency are still needed since even small improvements could positively influence the sustainability of vector production, the number of patients to be treated with each batch of vector as well as contribute to avoid triggering of innate sensor and effectors. A potential strategy to overcome innate barriers to transduction is the use of Cyclosporin A (CsA), an immuno-modulatory compound commonly used for the prevention of graft rejection, which is also known to significantly improve LV gene transfer in human and murine HSPC. This effect is cell specific and opposite to its well documented inhibitory effect on HIV replication. Tracking of the LV genome fate in treated HSPCs revealed that CsA relieves not-fully understood viral early block and increases integration. Hereby in this thesis the attention will be focused on the identification of several cellular partner candidates of CsA-mediated effect and the investigation of their role in LV restriction. Different set of genes were selected according to several criteria involving either direct CsA-interaction or based on specific RNAseq screening. In this context we produced HIV-1 derived vectors in order to over-express our genes of interest in K562 and THP-1 cell lines. The latter have been then transduced either with a second LV or with an integrase defective lentiviral vector (IDLV) to evaluate the impact of the over-expression on the transduction efficiency.