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Background: Cellular biobanks are of great interest for performing studies finalized in the development of personalized approaches for genetic diseases, including β-thalassemia and sickle cell disease (SCD), important diseases affecting the hematopoietic system. These inherited genetic diseases are characterized by a global distribution and the need for intensive health care. The aim of this report is to present an update on the composition of a cellular Thal-Biobank, to describe its utilization since 2016, to present data on its application in studies on fetal hemoglobin induction and on gene editing, and to discuss its employment as a "unique tool" during and after the COVID-19 pandemic. Methods: The methods were as follows: freezing, cryopreservation, long-term storage, and thawing of erythroid precursor cells from β-thalassemia patients; fetal hemoglobin (HbF) induction; CRISPR-Cas9 gene editing; HPLC analysis of the hemoglobin pattern. Results: The updated version of the Thal-Biobank is a cellular repository constituted of 990 cryovials from 221 β-thalassemia patients; the phenotype (pattern of hemoglobin production) is maintained after long-term storage; fetal hemoglobin induction and CRISPR-Cas9 gene editing can be performed using biobanked cells. In representative experiments using an isoxazole derivative as HbF inducer, the HbF increased from 13.36% to more than 60%. Furthermore, in CRIPR/Cas9 gene editing, de novo production of HbA was obtained (42.7% with respect to the trace amounts found in untreated cells). Conclusions: The implemented Thal-Biobank was developed before the COVID-19 outbreak and should be considered a tool of great interest for researchers working on β-thalassemia, with the aim of developing innovative therapeutic protocols and verifying the impact of the COVID-19 pandemic on erythroid precursor cells. [ABSTRACT FROM AUTHOR]

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from β-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for β-thalassemia. [ABSTRACT FROM AUTHOR]

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β‐Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in β‐Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN‐γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4+ and CD8+ T cells, respectively. The main results of the present study are that treatment of β‐Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4+ and CD8+ T cells releasing IFN‐γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β‐Thalassemia patients are considered prone to immune deficiency. [ABSTRACT FROM AUTHOR]

Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF. [ABSTRACT FROM AUTHOR]