Gene editing by the CRISPR-Cas9 nuclease system technology can be considered among the most promising strategies to correct hereditary mutations in a variety of monogenic diseases. In this paper, we present for the first time the correction, by CRISPR-Cas9 gene editing, of the β⁰39-thalassemia mutation, one of the most frequent in the Mediterranean area. The results obtained demonstrated the presence of normal β-globin genes after CRISPR-Cas9 correction of the β⁰39-thalassemia mutation performed on erythroid precursor cells from homozygous β⁰39-thalassemia patients. This was demonstrated by allele-specific PCR and sequencing. Accumulation of corrected β-globin mRNA and relevant “de novo” production of β-globin and adult hemoglobin (HbA) were found with high efficiency. The CRISPR-Cas9-forced HbA production levels were associated with a significant reduction of the excess of free α-globin chains. Genomic toxicity of the editing procedure (low indels and no off-targeting) was analyzed. The protocol might be the starting point for the development of an efficient editing of CD34⁺ cells derived from β⁰39 patients and for the design of combined treatments using, together with the CRISPR-Cas9 editing of the β-globin gene, other therapeutic approaches, such as, for instance, induction of HbA and/or fetal hemoglobin (HbF) using chemical inducers.

The present study investigated the effects of the combined treatment of two peptide nucleic acids (PNAs), directed against microRNAs involved in caspase‑3 mRNA regulation (miR‑155‑5p and miR‑221‑3p) in the temozolomide (TMZ)‑resistant T98G glioma cell line. These PNAs were conjugated with an octaarginine tail in order to obtain an efficient delivery to treated cells. The effects of singularly administered PNAs or a combined treatment with both PNAs were examined on apoptosis, with the aim to determine whether reversion of the drug‑resistance phenotype was obtained. Specificity of the PNA‑mediated effects was analyzed by reverse transcription‑quantitative polymerase‑chain reaction, which demonstrated that the effects of R8‑PNA‑a155 and R8-PNA-a221 anti‑miR PNAs were specific. Furthermore, the results obtained confirmed that both PNAs induced apoptosis when used on the temozolomide‑resistant T98G glioma cell line. Notably, co‑administration of both anti‑miR‑155 and anti‑miR‑221 PNAs was associated with an increased proapoptotic activity. In addition, TMZ further increased the induction of apoptosis in T98G cells co‑treated with anti‑miR‑155 and anti‑miR‑221 PNAs.

Introduction: β-thalassemia is caused by autosomal mutations in the β-globin gene, which induce the absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with β-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus that serves as a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for β-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin). Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitro cultured erythroid precursors). Results: The results were obtained in eight patients with the β⁺/β⁺ and β⁺/β⁰ genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24–48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis. Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with β-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies.

Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on β-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. Erythroid precursor cells were obtained from 72 patients, mostly β-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from β-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.

β-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding β-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for β-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from β-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine β-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of β-thalassemia.

• Optical biosensor for sickle cell anemia diagnostics. • SPR-based biosensor to identify single point mutations. • Reliable and reproducible detection of βS mutation.