Paolo Moi - Profile on Academia.edu (original) (raw)
Papers by Paolo Moi
HPFH from Klf1 haploinsufficiency combined to transcriptional heterozygous b-thalassemia
Normal Δ-Globin Gene Sequences in Sardinian Nondeletional Δβ-Thalassemia
Hemoglobin, 1992
ABSTRACT In order to clarify the reasons for the reduced Hb A2 levels in Sardinian δβ-thalassemia... more ABSTRACT In order to clarify the reasons for the reduced Hb A2 levels in Sardinian δβ-thalassemia, we characterized, both by cloning and sequence analysis and by direct sequencing of amplified DNA, the δ-globin gene from an individual of Sardinian descent who is a compound heterozygote for the β β-thalassemia codon 39 (C→T) nonsense mutation and the Sardinian δβ-thalassemia [codon 39(C→T)/ -196(C→T)Aγ]. The analysis of the δ-globin gene from the δβ-thal-assemia chromosome revealed an entirely normal sequence. The defective function of the δ-globin gene in this determinant is thus likely related to a suppressive effect of the in cis nondele-tional high persistence of fetal hemoglobin mutation of the Aγ gene, probably resulting from an increased capability of the relative promoter to interact with the locus control region.
Journal of Clinical Investigation, Nov 1, 1987
a-globin is encoded by two adjacent genes, al and a2. Recent evidence suggests that these genes a... more a-globin is encoded by two adjacent genes, al and a2. Recent evidence suggests that these genes are not equally expressed and that the a2-globin gene encodes the majority of a-globin. This finding would predict that a thalassemic mutation of the a2-globin gene would result in a more severe loss of a-chain synthesis than a similar mutation in the al-globin gene. In a previous study we described a nondeletion a-thalassemia de- fect in the a2-globin gene resulting from an AUG --ACG initiation codon mutation. In the present study we describe a different initiation codon mutation, AUG -GUG, present in the al-globin gene. The al-and a2-globin gene initiation codon mutations result in similarly lowered levels of encoded mRNA. Despite the similarity of these two mutations, the a2 mutant results in a more severe loss of a-globin synthesis and a more severe clinical a-thalassemia phenotype than the corre- sponding al-globin gene mutation. This difference reflects the dominant role of a2-globin gene in overall a-globin synthesis.
Regulation of the Human α-Globin Genes by GKLF
Blood, Nov 16, 2008
EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β-... more EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β- and β-like globin genes. Prompted by the observation that four KLF sites are distributed in the human α-globin promoter, we investigated if any of the β-globin cluster regulating KLFs could also act to modulate the expression of the α-globin genes. We found that, among the globin regulating KLFs (EKLF, LKLF, BKLF, GKLF, KLF6, FKLF and FKLF2), only GKLF and BKLF bound specifically to three out of four KLF sites. In K562 cells, over-expressed GKLF transactivated at high levels a α-globin-luciferase reporter and its action was impaired by point mutations of the KLF sites that disrupted GKLFDNA binding. In K562 cells stably transfected with a Tet-off regulated GKLF expression cassette, GKLF induction stimulated the expression of the endogenous α-globin genes. In a complementary assay in K562 cells, knocking down GKLF expression with small interfering RNAs caused a parallel decrease in the transcription of the α-globin genes. All experiments combined support a main regulatory role of GKLF in the control of α-globin gene expression.
Genetic modifier factors of ß-thalassemia [Modificatori genetici della ß-talassemia]
Area Pediatrica, 2011
Table 4. [Selected HBA1 and HBA2 Pathogenic Variants]
The distalβ-globin CACCC box is required for maximal stimulation of theβ-globin gene by EKLF
British Journal of Haematology, Sep 2, 2004
The transcription factor erythroid Kruppel-like factor (EKLF) specifically activates the beta-glo... more The transcription factor erythroid Kruppel-like factor (EKLF) specifically activates the beta-globin gene by interacting with the proximal beta-globin CACCC box, a known hot spot for thalassaemia mutations. This study investigated whether EKLF could also bind to, and activate from, the distal CACCC, which is a rare site of thalassaemia mutations. Using band shift and transient expression analysis with wild type, single and double CACCC mutants, we established that the distal CACCC box is weakly bound by EKLF, but, when mutated, significantly impairs EKLF-dependent beta-globin stimulation. Thus, EKLF requires both CACCC boxes to maximally stimulate the beta-globin gene.
Figure 2. [Diagram of the α-globin gene...]
Figure 1. [Diagram of the α-globin gene...]
Table 3. [Summary of Molecular Genetic Testing Used in Alpha-Thalassemia]
Figure 2. [Diagnostic algorithm for hemoglobinopathies]
Low Impact of Genetic Modifiers on the Phenotype of Homozygous Beta Thalassemia in the Last Decennial Cohort of Thalassemia Newborns in Sardinia
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>
MOESM1 of A validated cellular biobank for β-thalassemia
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.
Lentiviral globin gene therapy with reduced-intensity conditioning in adults with β-thalassemia: a phase 1 trial
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.
Evidence for Three Distinct Classes of Phenotype Severity in Beta-Thalassaemia
SSRN Electronic Journal, 2019
The multicenter THALAMOSS cellular Biobank for β-Thalassemia
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
Cloning, characterization, DNA binding, mapping and mutation analysis of the human erythroid kruppel-like factor (EKLF) gene
Figure 2. [Diagnostic algorithm for hemoglobinopathies]
Table 6. [Selected HBA1 and HBA2 Pathogenic Variants]
Table 1. [Red Blood Cell Indices in Adults with Alpha-Thalassemia]
HPFH from Klf1 haploinsufficiency combined to transcriptional heterozygous b-thalassemia
Normal Δ-Globin Gene Sequences in Sardinian Nondeletional Δβ-Thalassemia
Hemoglobin, 1992
ABSTRACT In order to clarify the reasons for the reduced Hb A2 levels in Sardinian δβ-thalassemia... more ABSTRACT In order to clarify the reasons for the reduced Hb A2 levels in Sardinian δβ-thalassemia, we characterized, both by cloning and sequence analysis and by direct sequencing of amplified DNA, the δ-globin gene from an individual of Sardinian descent who is a compound heterozygote for the β β-thalassemia codon 39 (C→T) nonsense mutation and the Sardinian δβ-thalassemia [codon 39(C→T)/ -196(C→T)Aγ]. The analysis of the δ-globin gene from the δβ-thal-assemia chromosome revealed an entirely normal sequence. The defective function of the δ-globin gene in this determinant is thus likely related to a suppressive effect of the in cis nondele-tional high persistence of fetal hemoglobin mutation of the Aγ gene, probably resulting from an increased capability of the relative promoter to interact with the locus control region.
Journal of Clinical Investigation, Nov 1, 1987
a-globin is encoded by two adjacent genes, al and a2. Recent evidence suggests that these genes a... more a-globin is encoded by two adjacent genes, al and a2. Recent evidence suggests that these genes are not equally expressed and that the a2-globin gene encodes the majority of a-globin. This finding would predict that a thalassemic mutation of the a2-globin gene would result in a more severe loss of a-chain synthesis than a similar mutation in the al-globin gene. In a previous study we described a nondeletion a-thalassemia de- fect in the a2-globin gene resulting from an AUG --ACG initiation codon mutation. In the present study we describe a different initiation codon mutation, AUG -GUG, present in the al-globin gene. The al-and a2-globin gene initiation codon mutations result in similarly lowered levels of encoded mRNA. Despite the similarity of these two mutations, the a2 mutant results in a more severe loss of a-globin synthesis and a more severe clinical a-thalassemia phenotype than the corre- sponding al-globin gene mutation. This difference reflects the dominant role of a2-globin gene in overall a-globin synthesis.
Regulation of the Human α-Globin Genes by GKLF
Blood, Nov 16, 2008
EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β-... more EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β- and β-like globin genes. Prompted by the observation that four KLF sites are distributed in the human α-globin promoter, we investigated if any of the β-globin cluster regulating KLFs could also act to modulate the expression of the α-globin genes. We found that, among the globin regulating KLFs (EKLF, LKLF, BKLF, GKLF, KLF6, FKLF and FKLF2), only GKLF and BKLF bound specifically to three out of four KLF sites. In K562 cells, over-expressed GKLF transactivated at high levels a α-globin-luciferase reporter and its action was impaired by point mutations of the KLF sites that disrupted GKLFDNA binding. In K562 cells stably transfected with a Tet-off regulated GKLF expression cassette, GKLF induction stimulated the expression of the endogenous α-globin genes. In a complementary assay in K562 cells, knocking down GKLF expression with small interfering RNAs caused a parallel decrease in the transcription of the α-globin genes. All experiments combined support a main regulatory role of GKLF in the control of α-globin gene expression.
Genetic modifier factors of ß-thalassemia [Modificatori genetici della ß-talassemia]
Area Pediatrica, 2011
Table 4. [Selected HBA1 and HBA2 Pathogenic Variants]
The distalβ-globin CACCC box is required for maximal stimulation of theβ-globin gene by EKLF
British Journal of Haematology, Sep 2, 2004
The transcription factor erythroid Kruppel-like factor (EKLF) specifically activates the beta-glo... more The transcription factor erythroid Kruppel-like factor (EKLF) specifically activates the beta-globin gene by interacting with the proximal beta-globin CACCC box, a known hot spot for thalassaemia mutations. This study investigated whether EKLF could also bind to, and activate from, the distal CACCC, which is a rare site of thalassaemia mutations. Using band shift and transient expression analysis with wild type, single and double CACCC mutants, we established that the distal CACCC box is weakly bound by EKLF, but, when mutated, significantly impairs EKLF-dependent beta-globin stimulation. Thus, EKLF requires both CACCC boxes to maximally stimulate the beta-globin gene.
Figure 2. [Diagram of the α-globin gene...]
Figure 1. [Diagram of the α-globin gene...]
Table 3. [Summary of Molecular Genetic Testing Used in Alpha-Thalassemia]
Figure 2. [Diagnostic algorithm for hemoglobinopathies]
Low Impact of Genetic Modifiers on the Phenotype of Homozygous Beta Thalassemia in the Last Decennial Cohort of Thalassemia Newborns in Sardinia
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>
MOESM1 of A validated cellular biobank for β-thalassemia
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.
Lentiviral globin gene therapy with reduced-intensity conditioning in adults with β-thalassemia: a phase 1 trial
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.
Evidence for Three Distinct Classes of Phenotype Severity in Beta-Thalassaemia
SSRN Electronic Journal, 2019
The multicenter THALAMOSS cellular Biobank for β-Thalassemia
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
Cloning, characterization, DNA binding, mapping and mutation analysis of the human erythroid kruppel-like factor (EKLF) gene
Figure 2. [Diagnostic algorithm for hemoglobinopathies]
Table 6. [Selected HBA1 and HBA2 Pathogenic Variants]
Table 1. [Red Blood Cell Indices in Adults with Alpha-Thalassemia]