Paolo Moi - Academia.edu (original) (raw)
Papers by Paolo Moi
Blood, Dec 7, 2017
<jats:title>Abstract</jats:title> <jats:p>Historically 15-20% of homozygous ß-t... more <jats:title>Abstract</jats:title> <jats:p>Historically 15-20% of homozygous ß-thalassemia in Sardinia developed the phenotype of thalassemia intermedia not requiring transfusion for survival. However, this category of patients has broad and heterogeneous spectrum severity, embracing truly mild thalassemia patients and patients with severity similar to thalassemia major. Known genetic modifiers that ameliorate the severity of b-thalassemia are the mild genotypes of ß+-thalassemia, the coinheritance of α-thalassemia, of hereditary persistence of fetal hemoglobin (HPFH) and of polymorphic variants in the BCL11a, MYB and HBG1 genes. All together this modifiers allow the calculation of a genetic score of severity that predicts the possibility of remaining transfusion free at a given age, likely aiding in the clinical decision to start or delay the initiation of a stable transfusion program. In this study we planned to evaluate the ameliorating effects of these genetic modifiers on the thalassemia phenotype by prospectively evaluating 51 homozygous ß-thalassemia newborns followed at our Institution in the last ten years. All newborns were genotyped soon after birth for all known genetic modifiers and monitored clinically for the appearance of anemia or other complications with monthly check-ups from birth until they required transfusions, according to the TIF guidelines. In this decennial cohort, all but 2 patients younger than 20 months started a regular transfusion program before 36 months of age. Hence, the percentage of thalassemia intermedia in Sardinia has dropped in the last decade from 18 to 4% and might even reach 0% if the 2 patients non yet transfused will transfuse within the next year. This shift from thalassemia intermedia to major is mainly accounted for by the great improvement in mortality, morbility and quality of life for thalassemia major compared to thalassemia intermedia patients who frequently develop difficult to manage complications with advancing age. In the cohort, the reason for start of blood transfusions was anemia lower than 7gr/dl in 14 % of cases and the appearance of skeletal modifications or growth failure in the remaining 84% of patients. Thus, differently from the past, most patients were transfused despite an acceptable grade of anemia. As expected, the presence of a greater number of genetic modifiers significantly correlated with a delay in the time lag to the first transfusion. Even better positive correlation (p&lt;0.0009) was found between the number of genetic modifiers and the levels of hemoglobin before beginning transfusion, confirming the positive influence of genetic modifiers on hemoglobin production. Since the start of transfusions, blood consumption did not positively correlated with the number of favorable genetic hits, but showed a trend toward significance, likely to achieve significance with increasing number of patients. Since the clinical decision to transfuse was taken mainly on the highly subjective parameter of appearance of skeletal modifications, it is possible that were transfused even the patients likely to have mild thalassemia intermedia and a good quality of life for most of their lifespan. We propose that at least the thalassemia patients with the best hemoglobin values (higher than 9 gr/dl) and a favorable background with at least 2 genetic modifiers (12% of our ߰-thalassemia newborns) should be more carefully evaluated for the possibility to evolve into a mild thalassemia intermedia phenotype with good quality of life for most of their lifespan.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec>
Additional file 1: Table S1. List of the subjects (patients and healthy subjects) present in the ... more Additional file 1: Table S1. List of the subjects (patients and healthy subjects) present in the Thal-Biobank.
Nature Medicine, 2022
β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of ... more β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the β chain of hemoglobin. Here we report 6–8-year follow-up of four adult patients with transfusion-dependent β-thalassemia who were infused with autologous CD34 + cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC—primary endpoint) and engraftment of genetically modified autologous CD34 + cells, expression of the transduced β-globin gene and post-transplant transfusion requirements (efficacy—secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34 + cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors. A gene therapy phase 1 trial in patients with β-thalassemia shows transplantation of autologous CD34 + cells transduced with a lentiviral globin vector after reduced-intensity conditioning achieves long-term engraftment, albeit not transfusion independence, with benign clonal expansions, warranting cautious monitoring of patients.
SSRN Electronic Journal, 2019
International Journal of Molecular Medicine, 2016
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 (THalassemia MOdular Stratification System for personalized therapy of β-thalassemia). The cellular Biobank contains hematopoietic stem cells isolated from the peripheral blood, expanded, freezed and cryopreserved. The use of biobanked samples allows to compare potentially therapeutic treatments on several samples growing in parallel. Freezing, cryopreservation and thawing steps do not affect the erythroid differentiation potential of the cells in terms of kinetics and types of hemoglobin produced by the cells obtained from different biobanked cryovials of the same patient and thawed with intervals reaching several months. The validation the cellular Biobank was further performed by sending and culturing the crypreserved cells from the same patients in different laboratories. Finally, we demonstrated that the biobanked cells can be used for determining the response to different inducers, such as mithramycin, hydroxyurea and resveratrol. Moreover, the biobanked cells can be used to test their response to gene therapy protocols. At present, the THALAMOSS cellular Biobank is composed by samples from 135 β-thalassemia patients and 19 healthy donors for a totale of more than 750 cryovials. The most represented genotypes are β039/β039 (51 patients), β+IVSI-6/β+IVSI-110 (31 patients), β039/β+IVSI-110 (21 patients) and β+IVSI-110/β+IVSI-110 (9 patients). In conclusion, this research activity will allow patients stratification taking into account all the phenotypic/genotypic characteristics of the single individual, and the response to potential therapeutic treatments
Blood, 2005
The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globi... more The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globin genes. While the β-globin CACCC box binds EKLF/KLF1, a likely adult switching factor, analogous factors, interacting with the γ-globin gene and predicted to regulate the fetal stage of hemoglobin switching, have so far been elusive. By using yeast one hybrid assay, we have isolated four KLFs, KLF1, 2, 4, and 6, that bound the γ-CACCC bait. To establish their role in globin regulation and in the switching of hemoglobins, these factors were compared to four other KLFs already established or putative globin regulators, KLF3, 11, 13 and 16, mainly evaluating their ability to bind and transactivate the ε-, γ- and β-globin gene. γ-CACCC binding at variable intensities was confirmed in band shift assay for all four isolated KLFs, for KLF3 and, faintly, for KLF13. The ε- and β-CACCC were bound by the same factors with similar affinities with the exception of KLF3 and KLF13 that bound stronger ...
Blood, 2005
The δ globin gene is the second adult β-like globin gene in humans and codes for the δ globin cha... more The δ globin gene is the second adult β-like globin gene in humans and codes for the δ globin chain which forms together with the α globin chain Hemoglobin A2 (HbA2). HbA2 represents less than 3% of the total hemoglobin in normal individuals and it is typically increased in β thalassemia carriers. The δ globin gene is highly homologous to the β globin gene since it derives from a common ancestor. In our previous work we (as well as others) have demonstrated, in vitro, that the creation of the β globin proximal CACCC box consensus sequence, the binding site of the trascription factor EKLF, on the δ globin gene promoter is sufficient to enhance its expression to a considerable extent. Here we show that the δ globin gene promoter can be activated “ in vivo” in a transgenic mice model. We have produced transgenic mice lines with a DNA construct in which the wild type (wt) β globin gene promoter and either the wt or the proximal CACCC box containing δ globin gene promoter are linked in c...
Blood, 2013
To date, the only curative therapeutic approach for beta-thalassemia major has been allogeneic st... more To date, the only curative therapeutic approach for beta-thalassemia major has been allogeneic stem cell transplantation (SCT) for patients with HLA-matched siblings. For the majority of patients who do not have a matched sibling, allogeneic SCT is associated with major risks of morbidity and mortality. The stable transfer of a functional globin gene into the patient’s own hematopoietic progenitor cells (HPCs) yields a perfectly matched graft that does not require immunosuppression to engraft. We previously demonstrated successful globin gene therapy in murine thalassemia models, using a lentiviral vector that encodes the human ß-globin promoter and arrayed regulatory elements uniquely combined to achieve high level and erythroid-specific globin expression. In vivo in thalassemic mice, the vector termed TNS9.3.55, increased hemoglobin levels by an average 4-6 g/dL per vector copy. We obtained in 2012 the first US Food and Drug Administration (FDA) approval to proceed to a clinical s...
Blood, 2011
2152 Hypersensitive site 2 (HS2) of the locus control region (LCR) is required for the optimal re... more 2152 Hypersensitive site 2 (HS2) of the locus control region (LCR) is required for the optimal regulation of the beta globin gene cluster. Screening a λgt11 cord blood cDNA library with the tandem NFE2 repeat of HS2 as recognition site probe, we isolated 14 cDNA clones of HMGB2, a chromatin non histone protein. Binding to the HS2 region was confirmed in vivo by ChIP assay. Transactivation analysis in K562 cells showed mild repression of a luciferase reporter driven by HS2 and the γ-promoter. The DNA bending capacity and the increased expression of HMGB2 during erythroid differentiation are properties well suited to facilitate LCR looping toward the β-globin genes, the mechanism thought to mediate globin gene activation. Hence, HMGB2 binding to HS2 may be relevant for the regulation of the β-globin gene cluster. To assess the function of HMGB2 as a possible regulator of the globin genes we analyzed the hematological phenotype of the HMGB2 knock out mice during erythroid differentiati...
Annals of Hematology, 2017
Blood, Dec 7, 2017
<jats:title>Abstract</jats:title> <jats:p>Historically 15-20% of homozygous ß-t... more <jats:title>Abstract</jats:title> <jats:p>Historically 15-20% of homozygous ß-thalassemia in Sardinia developed the phenotype of thalassemia intermedia not requiring transfusion for survival. However, this category of patients has broad and heterogeneous spectrum severity, embracing truly mild thalassemia patients and patients with severity similar to thalassemia major. Known genetic modifiers that ameliorate the severity of b-thalassemia are the mild genotypes of ß+-thalassemia, the coinheritance of α-thalassemia, of hereditary persistence of fetal hemoglobin (HPFH) and of polymorphic variants in the BCL11a, MYB and HBG1 genes. All together this modifiers allow the calculation of a genetic score of severity that predicts the possibility of remaining transfusion free at a given age, likely aiding in the clinical decision to start or delay the initiation of a stable transfusion program. In this study we planned to evaluate the ameliorating effects of these genetic modifiers on the thalassemia phenotype by prospectively evaluating 51 homozygous ß-thalassemia newborns followed at our Institution in the last ten years. All newborns were genotyped soon after birth for all known genetic modifiers and monitored clinically for the appearance of anemia or other complications with monthly check-ups from birth until they required transfusions, according to the TIF guidelines. In this decennial cohort, all but 2 patients younger than 20 months started a regular transfusion program before 36 months of age. Hence, the percentage of thalassemia intermedia in Sardinia has dropped in the last decade from 18 to 4% and might even reach 0% if the 2 patients non yet transfused will transfuse within the next year. This shift from thalassemia intermedia to major is mainly accounted for by the great improvement in mortality, morbility and quality of life for thalassemia major compared to thalassemia intermedia patients who frequently develop difficult to manage complications with advancing age. In the cohort, the reason for start of blood transfusions was anemia lower than 7gr/dl in 14 % of cases and the appearance of skeletal modifications or growth failure in the remaining 84% of patients. Thus, differently from the past, most patients were transfused despite an acceptable grade of anemia. As expected, the presence of a greater number of genetic modifiers significantly correlated with a delay in the time lag to the first transfusion. Even better positive correlation (p&lt;0.0009) was found between the number of genetic modifiers and the levels of hemoglobin before beginning transfusion, confirming the positive influence of genetic modifiers on hemoglobin production. Since the start of transfusions, blood consumption did not positively correlated with the number of favorable genetic hits, but showed a trend toward significance, likely to achieve significance with increasing number of patients. Since the clinical decision to transfuse was taken mainly on the highly subjective parameter of appearance of skeletal modifications, it is possible that were transfused even the patients likely to have mild thalassemia intermedia and a good quality of life for most of their lifespan. We propose that at least the thalassemia patients with the best hemoglobin values (higher than 9 gr/dl) and a favorable background with at least 2 genetic modifiers (12% of our ߰-thalassemia newborns) should be more carefully evaluated for the possibility to evolve into a mild thalassemia intermedia phenotype with good quality of life for most of their lifespan.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>No relevant conflicts of interest to declare.</jats:p> </jats:sec>
Additional file 1: Table S1. List of the subjects (patients and healthy subjects) present in the ... more Additional file 1: Table S1. List of the subjects (patients and healthy subjects) present in the Thal-Biobank.
Nature Medicine, 2022
β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of ... more β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the β chain of hemoglobin. Here we report 6–8-year follow-up of four adult patients with transfusion-dependent β-thalassemia who were infused with autologous CD34 + cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC—primary endpoint) and engraftment of genetically modified autologous CD34 + cells, expression of the transduced β-globin gene and post-transplant transfusion requirements (efficacy—secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34 + cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors. A gene therapy phase 1 trial in patients with β-thalassemia shows transplantation of autologous CD34 + cells transduced with a lentiviral globin vector after reduced-intensity conditioning achieves long-term engraftment, albeit not transfusion independence, with benign clonal expansions, warranting cautious monitoring of patients.
SSRN Electronic Journal, 2019
International Journal of Molecular Medicine, 2016
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 (THalassemia MOdular Stratification System for personalized therapy of β-thalassemia). The cellular Biobank contains hematopoietic stem cells isolated from the peripheral blood, expanded, freezed and cryopreserved. The use of biobanked samples allows to compare potentially therapeutic treatments on several samples growing in parallel. Freezing, cryopreservation and thawing steps do not affect the erythroid differentiation potential of the cells in terms of kinetics and types of hemoglobin produced by the cells obtained from different biobanked cryovials of the same patient and thawed with intervals reaching several months. The validation the cellular Biobank was further performed by sending and culturing the crypreserved cells from the same patients in different laboratories. Finally, we demonstrated that the biobanked cells can be used for determining the response to different inducers, such as mithramycin, hydroxyurea and resveratrol. Moreover, the biobanked cells can be used to test their response to gene therapy protocols. At present, the THALAMOSS cellular Biobank is composed by samples from 135 β-thalassemia patients and 19 healthy donors for a totale of more than 750 cryovials. The most represented genotypes are β039/β039 (51 patients), β+IVSI-6/β+IVSI-110 (31 patients), β039/β+IVSI-110 (21 patients) and β+IVSI-110/β+IVSI-110 (9 patients). In conclusion, this research activity will allow patients stratification taking into account all the phenotypic/genotypic characteristics of the single individual, and the response to potential therapeutic treatments
Blood, 2005
The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globi... more The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globin genes. While the β-globin CACCC box binds EKLF/KLF1, a likely adult switching factor, analogous factors, interacting with the γ-globin gene and predicted to regulate the fetal stage of hemoglobin switching, have so far been elusive. By using yeast one hybrid assay, we have isolated four KLFs, KLF1, 2, 4, and 6, that bound the γ-CACCC bait. To establish their role in globin regulation and in the switching of hemoglobins, these factors were compared to four other KLFs already established or putative globin regulators, KLF3, 11, 13 and 16, mainly evaluating their ability to bind and transactivate the ε-, γ- and β-globin gene. γ-CACCC binding at variable intensities was confirmed in band shift assay for all four isolated KLFs, for KLF3 and, faintly, for KLF13. The ε- and β-CACCC were bound by the same factors with similar affinities with the exception of KLF3 and KLF13 that bound stronger ...
Blood, 2005
The δ globin gene is the second adult β-like globin gene in humans and codes for the δ globin cha... more The δ globin gene is the second adult β-like globin gene in humans and codes for the δ globin chain which forms together with the α globin chain Hemoglobin A2 (HbA2). HbA2 represents less than 3% of the total hemoglobin in normal individuals and it is typically increased in β thalassemia carriers. The δ globin gene is highly homologous to the β globin gene since it derives from a common ancestor. In our previous work we (as well as others) have demonstrated, in vitro, that the creation of the β globin proximal CACCC box consensus sequence, the binding site of the trascription factor EKLF, on the δ globin gene promoter is sufficient to enhance its expression to a considerable extent. Here we show that the δ globin gene promoter can be activated “ in vivo” in a transgenic mice model. We have produced transgenic mice lines with a DNA construct in which the wild type (wt) β globin gene promoter and either the wt or the proximal CACCC box containing δ globin gene promoter are linked in c...
Blood, 2013
To date, the only curative therapeutic approach for beta-thalassemia major has been allogeneic st... more To date, the only curative therapeutic approach for beta-thalassemia major has been allogeneic stem cell transplantation (SCT) for patients with HLA-matched siblings. For the majority of patients who do not have a matched sibling, allogeneic SCT is associated with major risks of morbidity and mortality. The stable transfer of a functional globin gene into the patient’s own hematopoietic progenitor cells (HPCs) yields a perfectly matched graft that does not require immunosuppression to engraft. We previously demonstrated successful globin gene therapy in murine thalassemia models, using a lentiviral vector that encodes the human ß-globin promoter and arrayed regulatory elements uniquely combined to achieve high level and erythroid-specific globin expression. In vivo in thalassemic mice, the vector termed TNS9.3.55, increased hemoglobin levels by an average 4-6 g/dL per vector copy. We obtained in 2012 the first US Food and Drug Administration (FDA) approval to proceed to a clinical s...
Blood, 2011
2152 Hypersensitive site 2 (HS2) of the locus control region (LCR) is required for the optimal re... more 2152 Hypersensitive site 2 (HS2) of the locus control region (LCR) is required for the optimal regulation of the beta globin gene cluster. Screening a λgt11 cord blood cDNA library with the tandem NFE2 repeat of HS2 as recognition site probe, we isolated 14 cDNA clones of HMGB2, a chromatin non histone protein. Binding to the HS2 region was confirmed in vivo by ChIP assay. Transactivation analysis in K562 cells showed mild repression of a luciferase reporter driven by HS2 and the γ-promoter. The DNA bending capacity and the increased expression of HMGB2 during erythroid differentiation are properties well suited to facilitate LCR looping toward the β-globin genes, the mechanism thought to mediate globin gene activation. Hence, HMGB2 binding to HS2 may be relevant for the regulation of the β-globin gene cluster. To assess the function of HMGB2 as a possible regulator of the globin genes we analyzed the hematological phenotype of the HMGB2 knock out mice during erythroid differentiati...
Annals of Hematology, 2017