In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to... more In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to induce γ-globin gene expression and increased production of fetal hemoglobin (HbF) in erythroid cells, including primary erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. For this reason, rapamycin is considered of great interest for the treatment of β-thalassemia. In fact, high levels of HbF are known to be highly beneficial for β-thalassemia patients. The story of rapamycin discovery began in 1964, with METEI, the Medical Expedition to Easter Island (Rapa Nui). During this expedition, samples of the soil from different parts of the island were collected and, from this material, an antibiotic-producing microorganism (Streptomyces hygroscopicus) was identified. Rapamycin was extracted from the mycelium with organic solvents, isolated, and demonstrated to be very active as an anti-bacterial and anti-fungal agent. Later, rapamycin was demonstrated to inhibit the in v...
The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regu... more The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5′-GGTTAT-3′) is located within the 5′-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in β-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify β-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in β-thalassemia patients. The most striking association was found using rapamycin as a...
One of the most relevant pathophysiological hallmarks of β-thalassemia is the accumulation of tox... more One of the most relevant pathophysiological hallmarks of β-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the...
The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production ... more The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the β-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α...
The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-1... more The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extrac...
The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding t... more The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from β-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of β-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that ...
Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic heredita... more 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 in...
Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Dr... more Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Drug repositioning and similar terms ('drug repurposing', 'drug reprofiling', 'drug redirecting', ‘drug rescue’, ‘drug re-tasking’ and/or 'drug rediscovery') have gained great attention, especially in the field or rare diseases (RDs), and represent relevant novel drug development strategies to be considered together with the “off-label” use of pharmaceutical products under clinical trial regimen. The most significant advantage of drug repositioning over traditional drug development is that the repositioned drug has already passed a significant number of short- and long-term toxicity tests, as well as it has already undergone pharmacokinetic and pharmacodynamic (PK/PD) studies. The established safety of repositioned drugs is known to significantly reduce the probability of project failure. Furthermore, development of repurposed drugs can shorten much of the t...
β-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the resul... more 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 ...
20th World Congress on Advances in Oncology and 18th International Symposium on Molecular Medicine, 2015
The production and validation of a cellular thalassemia biobank was conducted within the multicen... more The production and validation of a cellular thalassemia biobank was conducted within the multicenter European Project THALAMOSS (THALAssaemia MOdular Stratification System for personalized therapy of β-thalassemia). The cellular biobank contains hematopoietic stem cells isolated from peripheral blood, expanded and cryopreserved. This approach is based on: (a) the recruitment of β-thalassemia patients characterized for the genotype and possible fetal hemoglobin (HbF)-associated polymorphisms (XmnI rs7482144, BCL11A rs 1427407 and rs10189857, MYB rs9399137); (b) the set up of protocols to efficiently isolate, culture, freeze and thaw the hematopoietic stem cells from 25 ml aliquot of blood collected before transfusion; (c) the use of cultures for determining the effects of HbF inducers as well as the efficacy of gene therapy approaches. We demonstrated that freezing, cryopreservation and thawing steps do not affect the erythroid differentiation potential of the cells in terms of kinetics and types of hemoglobin produced. The validation of the cellular biobank was further performed by sending and culturing the cryopreserved cells from the same patients in different laboratories. Finally, we analyzed the response to different HbF inducers. The cells stored in the biobank were found to be responsive to mithramycin, hydroxyurea and resveratrol. At present, this first THALAMOSS cellular biobank is composed of 749 cryovials from 75 β-thalassemia patients. In conclusion, this research activity will allow patient stratification taking into account all the phenotypic/genotypic characteristics of the single individual associated with in vitro HbF induction under treatment with effective inducers, providing an important opportunity for the development of novel therapeutic strategies for β-thalassemia
The protocols here described are related to the sections presented in Figure 1B focusing on the p... more The protocols here described are related to the sections presented in Figure 1B focusing on the practical activity performed by the students. All the steps are presented and critically commented when deemed useful. The reagents used during the preparation of these protocols are indicated as just an example, as all the reagents can be purchased from any company.
A series of new-generation TMA (4,6,4′-trimethyl angelicin) analogues was projected and synthetiz... more A series of new-generation TMA (4,6,4′-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4′-dimethyl-angelicin) and GY964 (4-phenyl-6,4′-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, t...
In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to... more In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to induce γ-globin gene expression and increased production of fetal hemoglobin (HbF) in erythroid cells, including primary erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. For this reason, rapamycin is considered of great interest for the treatment of β-thalassemia. In fact, high levels of HbF are known to be highly beneficial for β-thalassemia patients. The story of rapamycin discovery began in 1964, with METEI, the Medical Expedition to Easter Island (Rapa Nui). During this expedition, samples of the soil from different parts of the island were collected and, from this material, an antibiotic-producing microorganism (Streptomyces hygroscopicus) was identified. Rapamycin was extracted from the mycelium with organic solvents, isolated, and demonstrated to be very active as an anti-bacterial and anti-fungal agent. Later, rapamycin was demonstrated to inhibit the in v...
The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regu... more The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5′-GGTTAT-3′) is located within the 5′-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in β-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify β-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in β-thalassemia patients. The most striking association was found using rapamycin as a...
One of the most relevant pathophysiological hallmarks of β-thalassemia is the accumulation of tox... more One of the most relevant pathophysiological hallmarks of β-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the...
The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production ... more The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the β-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α...
The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-1... more The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extrac...
The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding t... more The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from β-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of β-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that ...
Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic heredita... more 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 in...
Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Dr... more Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Drug repositioning and similar terms ('drug repurposing', 'drug reprofiling', 'drug redirecting', ‘drug rescue’, ‘drug re-tasking’ and/or 'drug rediscovery') have gained great attention, especially in the field or rare diseases (RDs), and represent relevant novel drug development strategies to be considered together with the “off-label” use of pharmaceutical products under clinical trial regimen. The most significant advantage of drug repositioning over traditional drug development is that the repositioned drug has already passed a significant number of short- and long-term toxicity tests, as well as it has already undergone pharmacokinetic and pharmacodynamic (PK/PD) studies. The established safety of repositioned drugs is known to significantly reduce the probability of project failure. Furthermore, development of repurposed drugs can shorten much of the t...
β-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the resul... more 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 ...
20th World Congress on Advances in Oncology and 18th International Symposium on Molecular Medicine, 2015
The production and validation of a cellular thalassemia biobank was conducted within the multicen... more The production and validation of a cellular thalassemia biobank was conducted within the multicenter European Project THALAMOSS (THALAssaemia MOdular Stratification System for personalized therapy of β-thalassemia). The cellular biobank contains hematopoietic stem cells isolated from peripheral blood, expanded and cryopreserved. This approach is based on: (a) the recruitment of β-thalassemia patients characterized for the genotype and possible fetal hemoglobin (HbF)-associated polymorphisms (XmnI rs7482144, BCL11A rs 1427407 and rs10189857, MYB rs9399137); (b) the set up of protocols to efficiently isolate, culture, freeze and thaw the hematopoietic stem cells from 25 ml aliquot of blood collected before transfusion; (c) the use of cultures for determining the effects of HbF inducers as well as the efficacy of gene therapy approaches. We demonstrated that freezing, cryopreservation and thawing steps do not affect the erythroid differentiation potential of the cells in terms of kinetics and types of hemoglobin produced. The validation of the cellular biobank was further performed by sending and culturing the cryopreserved cells from the same patients in different laboratories. Finally, we analyzed the response to different HbF inducers. The cells stored in the biobank were found to be responsive to mithramycin, hydroxyurea and resveratrol. At present, this first THALAMOSS cellular biobank is composed of 749 cryovials from 75 β-thalassemia patients. In conclusion, this research activity will allow patient stratification taking into account all the phenotypic/genotypic characteristics of the single individual associated with in vitro HbF induction under treatment with effective inducers, providing an important opportunity for the development of novel therapeutic strategies for β-thalassemia
The protocols here described are related to the sections presented in Figure 1B focusing on the p... more The protocols here described are related to the sections presented in Figure 1B focusing on the practical activity performed by the students. All the steps are presented and critically commented when deemed useful. The reagents used during the preparation of these protocols are indicated as just an example, as all the reagents can be purchased from any company.
A series of new-generation TMA (4,6,4′-trimethyl angelicin) analogues was projected and synthetiz... more A series of new-generation TMA (4,6,4′-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4′-dimethyl-angelicin) and GY964 (4-phenyl-6,4′-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, t...
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Papers by Lucia Cosenza