Introduction: The β-thalassemias are due to autosomal mutations of the β-globin gene, inducing absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that high production of fetal hemoglobin (HbF) is beneficial for β-thalassemia patients. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus found to be a strong HbF inducer in vitro and in vivo. In this study, we report biochemical, molecular and clinical results of the 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 by reverse-transcription-quantitative PCR and the hemoglobin pattern by HPLC. The immunophenotype was analyzed by FACS using antibodies against CD3, CD4, CD8, CD14, CD19, CD25. Results: The results were obtained in 8 patients with β+/β+ and β+/β0 genotypes, treated with a starting dosage of 1 mg/day sirolimus for 24-48 weeks. The first finding of the study was that expression of γ-globin mRNA was increased in blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. A second important conclusion of our trial was that sirolimus influences erythropoiesis and reduces biochemical markers associated to ineffective erythropoiesis (I.E.) (excess of free α-globin chains, bilirubin, soluble transferrin receptor and ferritin). In most (7/8) of the patients a decrease of the transfusion index was observed. The drug was well tolerated with minor effects on immunophenotype, the only side effect being frequently occurring stomatitis. Conclusions: The data obtained indicate that sirolimus given at low doses modifies hematopoiesis and induces increased expression of γ-globin genes in a sub-set of β-thalassemia patients. Further clinical trials are warranted, considering the possibility to test the drug in patients with less severe forms of the disease and exploring combination therapies.

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 b 0 39-thalassemia mutation, one of the most frequent in the Mediterranean area. The results obtained demonstrated the presence of normal b-globin genes after CRISPR-Cas9 correction of the b 0 39-thalassemia mutation performed on erythroid precursor cells from homozygous b 0 39-thalassemia patients. This was demonstrated by allelespecific PCR and sequencing. Accumulation of corrected b-globin mRNA and relevant "de novo" production of b-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 a-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 b 0 39 patients and for the design of combined treatments using, together with the CRISPR-Cas9 editing of the b-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.

We found a novel polymorphism of the Aγ-globin gene in four families with β(0)-thalassemia and high levels of HbF expression. Additionally, we report evidence suggesting that the Aγ-globin gene +25(G→A) polymorphism decreases the efficiency of the interaction between this sequence and specific DNA binding protein complexes.