Cellular and biomolecular technologies for stratification of β thalassemia patients: applications in theranostics

The research described in the present PhD Thesis has been conducted in the context of a multicenter FP7 European Project called THALAMOSS (THalassemia MOdular Stratification System), having as major objective the identification of molecular markers for the development of personalized therapies for hemoglobinopathies, in particular ß-thalassemia. The ß-thalassemias are an autosomal recessive genetic disorders caused by the absence or reduction of ß-globin chains of adult hemoglobin, for which targeted and definitive treatments are, at present, not available. In order to sustain project based on stratification of ß-thalassemia patients according to clinical, genetic and molecular features, we established a systematic collection of cellular samples, the cellular Biobank, containing hematopoietic stem cells isolated from the peripheral blood, expanded, freezed and cryopreserved. To this aim, 75 subjects comprising ß-thalassemia patients and healthy donors have been recruited up to now. They were all characterized for the genotype (in order to detect pathogenic mutations) and possible fetal hemoglobin (HbF)-associated polymorphisms. More importantly, new protocols to efficiently isolate, culture, freeze and thaw hematopoietic stem cells were developed. In particular, we demonstrated that freezing, cryopreservation and thawing steps do not affect the erythroid differentiation potential of the cells and the natural erythroid differentiation process, in terms of kinetics and types of hemoglobin produced by the cells of the same patient. Moreover, we found that the cells stored in the Biobank are responsive, once thawed and sub-cultured, to treatments with known HbF inducers, including mithramycin, resveratrol, butyric acid and hydroxyurea, and are therefore suitable for the identification and development of new HbF inducers to be used for experimental therapeutic strategy for ß-thalassemia. In this context, we demonstrated that the induction effects depend on the subject’s genotype, strongly suggesting that this approach could be very useful to develop personalized therapies. In conclusion, this research activity will allow patients stratification taking into account all the phenotypic/genotypic characteristics of the single individual, in association with in vitro HbF induction under treatment with effective inducers, providing an important opportunity for the research and development of novel therapeutic strategies for ß-thalassemia.