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Professore Ordinario
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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2022 - Cerebellar Pathology in an Inducible Mouse Model of Friedreich Ataxia [Articolo su rivista]
Mercado-Ayon, E.; Warren, N.; Halawani, S.; Rodden, L. N.; Ngaba, L.; Dong, Y. N.; Chang, J. C.; Fonck, C.; Mavilio, F.; Lynch, D. R.; Lin, H.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by deficiency of the mitochondrial protein frataxin. Lack of frataxin causes neuronal loss in various areas of the CNS and PNS. In particular, cerebellar neuropathology in FRDA patients includes loss of large principal neurons and synaptic terminals in the dentate nucleus (DN), and previous studies have demonstrated early synaptic deficits in the Knockin-Knockout mouse model of FRDA. However, the exact correlation of frataxin deficiency with cerebellar neuropathology remains unclear. Here we report that doxycycline-induced frataxin knockdown in a mouse model of FRDA (FRDAkd) leads to synaptic cerebellar degeneration that can be partially reversed by AAV8-mediated frataxin restoration. Loss of cerebellar Purkinje neurons and large DN principal neurons are observed in the FRDAkd mouse cerebellum. Levels of the climbing fiber-specific glutamatergic synaptic marker VGLUT2 decline starting at 4 weeks after dox induction, whereas levels of the parallel fiber-specific synaptic marker VGLUT1 are reduced by 18-weeks. These findings suggest initial selective degeneration of climbing fiber synapses followed by loss of parallel fiber synapses. The GABAergic synaptic marker GAD65 progressively declined during dox induction in FRDAkd mice, while GAD67 levels remained unaltered, suggesting specific roles for frataxin in maintaining cerebellar synaptic integrity and function during adulthood. Expression of frataxin following AAV8-mediated gene transfer partially restored VGLUT1/2 levels. Taken together, our findings show that frataxin knockdown leads to cerebellar degeneration in the FRDAkd mouse model, suggesting that frataxin helps maintain cerebellar structure and function.

2022 - Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity [Articolo su rivista]
Eggers, M.; Vannoy, C. H.; Huang, J.; Purushothaman, P.; Brassard, J.; Fonck, C.; Meng, H.; Prom, M. J.; Lawlor, M. W.; Cunningham, J.; Sadhu, C.; Mavilio, F.

Pompe disease is a severe disorder caused by loss of acid α-glucosidase (GAA), leading to glycogen accumulation in tissues and neuromuscular and cardiac dysfunction. Enzyme replacement therapy is the only available treatment. AT845 is an adeno-associated viral vector designed to express human GAA specifically in skeletal muscle and heart. Systemic administration of AT845 in Gaa−/− mice led to a dose-dependent increase in GAA activity, glycogen clearance in muscles and heart, and functional improvement. AT845 was tolerated in cynomolgus macaques at low doses, while high doses caused anti-GAA immune response, inflammation, and cardiac abnormalities resulting in unscheduled euthanasia of two animals. Conversely, a vector expressing the macaque GAA caused no detectable pathology, indicating that the toxicity observed with AT845 was an anti-GAA xenogeneic immune response. Western blot analysis showed abnormal processing of human GAA in cynomolgus muscle, adding to the species-specific effects of enzyme expression. Overall, these studies show that AAV-mediated GAA delivery to muscle is efficacious in Gaa−/− mice and highlight limitations in predicting the toxicity of AAV vectors encoding human proteins in non-human species.

2021 - Clinical management of sickle cell liver disease in children and young adults [Articolo su rivista]
Kyrana, E.; Rees, D.; Lacaille, F.; Fitzpatrick, E.; Davenport, M.; Heaton, N.; Height, S.; Samyn, M.; Mavilio, F.; Brousse, V.; Suddle, A.; Chakravorty, S.; Verma, A.; Gupte, G.; Velangi, M.; Inusa, B.; Drasar, E.; Hadzic, N.; Grammatikopoulos, T.; Hind, J.; Deheragoda, M.; Sellars, M.; Dhawan, A.

Liver involvement in sickle cell disease (SCD) is often referred to as sickle cell hepatopathy (SCH) and is a complication of SCD which may be associated with significant mortality. This review is based on a round-table workshop between paediatric and adult hepatologists and haematologists and review of the literature. The discussion was prompted by the lack of substantial data and guidance in managing these sometimes very challenging cases. This review provides a structured approach for the diagnosis and management of SCH in children and young adults. The term SCH describes any hepatobiliary dysfunction in the context of SCD. Diagnosis and management of biliary complications, acute hepatic crisis, acute hepatic sequestration and other manifestations of SCH are discussed, as well as the role of liver transplantation and haemopoietic stem cell transplantation in the management of SCH.

2021 - Correction of b-thalassemia by CRISPR/Cas9 editing of the a-globin locus in human hematopoietic stem cells [Articolo su rivista]
Pavani, G.; Fabiano, A.; Laurent, M.; Amor, F.; Cantelli, E.; Chalumeau, A.; Maule, G.; Tachtsidi, A.; Concordet, J. -P.; Cereseto, A.; Mavilio, F.; Ferrari, G.; Miccio, A.; Amendola, M.

b-thalassemias (b-thal) are a group of blood disorders caused by mutations in the b-globin gene (HBB) cluster. b-globin associates with a-globin to form adult hemoglobin (HbA, a2b2), the main oxygen-carrier in erythrocytes. When b-globin chains are absent or limiting, free a-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of b-thal correlates with the number of inherited a-globin genes (HBA1 and HBA2), with a-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat b-thal based on genome editing of the a-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) a-globin downregulation, by deleting the HBA2 gene to recreate an a-thalassemia trait, and (2) b-globin expression, by targeted integration of a b-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of b-thalassemia (human erythroid progenitor cell [HUDEP-2] b0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited b-thal HSPCs and achieved correction of a/b globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve a/b globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for b-thal.

2021 - Designing lentiviral vectors for gene therapy of genetic diseases [Articolo su rivista]
Poletti, V.; Mavilio, F.

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, ob-tained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase.

2020 - Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype [Articolo su rivista]
Weber, L.; Frati, G.; Felix, T.; Hardouin, G.; Casini, A.; Wollenschlaeger, C.; Meneghini, V.; Masson, C.; de Cian, A.; Chalumeau, A.; Mavilio, F.; Amendola, M.; Andre-Schmutz, I.; Cereseto, A.; El Nemer, W.; Concordet, J. -P.; Giovannangeli, C.; Cavazzana, M.; Miccio, A.

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) β chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.

2020 - Erratum: Genomic analysis of sleeping beauty transposon integration in human somatic cells (PLoS ONE (2014) 9:11 (e112712) DOI: 10.1371/journal.pone.0112712) [Articolo su rivista]
Turchiano, G.; Latella, M. C.; Doring, A. G.; Cattoglio, C.; Mavilio, F.; Izsvak, Z.; Ivics, Z.; Recchia, A.

The following information is missing from the Funding statement: Dr. Zsuzsanna Izsvák was funded by European Research Council-2011-ADG-TRANSPOSOstress- 294742.

2019 - Biosafety Studies of a Clinically Applicable Lentiviral Vector for the Gene Therapy of Artemis-SCID [Articolo su rivista]
Charrier, S.; Lagresle-Peyrou, C.; Poletti, V.; Rothe, M.; Cedrone, G.; Gjata, B.; Mavilio, F.; Fischer, A.; Schambach, A.; de Villartay, J. -P.; Cavazzana, M.; Hacein-Bey-Abina, S.; Galy, A.

Genetic deficiency of the nuclease DCLRE1C/Artemis causes radiosensitive severe combined immunodeficiency (RS-SCID) with lack of peripheral T and B cells and increased sensitivity to ionizing radiations. Gene therapy based on transplanting autologous gene-modified hematopoietic stem cells could significantly improve the health of patients with RS-SCID by correcting their immune system. A lentiviral vector expressing physiological levels of human ARTEMIS mRNA from an EF1a promoter without post-transcriptional regulation was developed as a safe clinically applicable candidate for RS-SCID gene therapy. The vector was purified in GMP-comparable conditions and was not toxic in vitro or in vivo. Long-term engraftment of vector-transduced hematopoietic cells was achieved in irradiated Artemis-deficient mice following primary and secondary transplantation (6 months each). Vector-treated mice displayed T and B lymphopoiesis and polyclonal T cells, had structured lymphoid tissues, and produced immunoglobulins. Benign signs of inflammation were noted following secondary transplants, likely a feature of the model. There was no evidence of transgene toxicity and no induction of hematopoietic malignancy. In vitro, the vector had low genotoxic potential on murine hematopoietic progenitor cells using an immortalization assay. Altogether, these preclinical data show safety and efficacy, and support further development of the vector for the gene therapy of RS-SCID.

2019 - Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements [Articolo su rivista]
Lattanzi, Annalisa; Meneghini, Vasco; Pavani, Giulia; Amor, Fatima; Ramadier, Sophie; Felix, Tristan; Antoniani, Chiara; Masson, Cecile; Alibeu, Olivier; Lee, Ciaran; Porteus, Matthew H.; Bao, Gang; Amendola, Mario; Mavilio, Fulvio; Miccio, Annarita

Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9+ HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.

2018 - An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle Cell Disease Phenotype [Articolo su rivista]
Weber, L.; Poletti, V.; Magrin, E.; Antoniani, C.; Martin, S.; Bayard, C.; Sadek, H.; Felix, T.; Meneghini, V.; Antoniou, M. N.; El-Nemer, W.; Mavilio, F.; Cavazzana, M.; Andre-Schmutz, I.; Miccio, A.

Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling HBB variant is a potential treatment for sickle cell disease (SCD). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling HBB transgene (HBBAS3), a minimal HBB promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCD patients were transduced with LVs containing either HS2 and HS3 (β-AS3) or HS2, HS3, and HS4 (β-AS3 HS4). The inclusion of the HS4 element drastically reduced vector titer and infectivity in HSPCs, with negligible improvement of transgene expression. Conversely, the LV containing only HS2 and HS3 was able to efficiently transduce SCD bone marrow and Plerixafor-mobilized HSPCs, with anti-sickling HBB representing up to ∼60% of the total HBB-like chains. The expression of the anti-sickling HBB and the reduced incorporation of the βS-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling HBB transgene ameliorating the SCD cell phenotype.

2018 - Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors [Articolo su rivista]
Holstein, M.; Mesa-Nunez, C.; Miskey, C.; Almarza, E.; Poletti, V.; Schmeer, M.; Grueso, E.; Ordonez Flores, J. C.; Kobelt, D.; Walther, W.; Aneja, M. K.; Geiger, J.; Bonig, H. B.; Izsvak, Z.; Schleef, M.; Rudolph, C.; Mavilio, F.; Bueren, J. A.; Guenechea, G.; Ivics, Z.

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34 + cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34 + cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34 + cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol. Ivics and collegues refined the Sleeping Beauty transposon system for gene transfer in human hematopoietic stem and progenitor cells by vectorizing the transposon components as minicircle DNA and synthetic mRNA. The advanced vector system enables efficient and safe non-viral engineering of hematopoietic cells that can be transplanted into immunodeficient mice.

2018 - Ex Vivo COL7A1 Correction for Recessive Dystrophic Epidermolysis Bullosa Using CRISPR/Cas9 and Homology-Directed Repair [Articolo su rivista]
Izmiryan, A.; Ganier, C.; Bovolenta, M.; Schmitt, A.; Mavilio, F.; Hovnanian, A.

Recessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.

2018 - Gene Therapy for Hemoglobinopathies [Articolo su rivista]
Cavazzana, M.; Mavilio, F.

Gene therapy for β-Thalassemia and sickle-cell disease is based on transplantation of genetically corrected, autologous hematopoietic stem cells. Preclinical and clinical studies have shown the safety and efficacy of this therapeutic approach, currently based on lentiviral vectors to transfer a β-globin gene under the transcriptional control of regulatory elements of the β-globin locus. Nevertheless, a number of factors are still limiting its efficacy, such as limited stem-cell dose and quality, suboptimal gene transfer efficiency and gene expression levels, and toxicity of myeloablative regimens. In addition, the cost and complexity of the current vector and cell manufacturing clearly limits its application to patients living in less favored countries, where hemoglobinopathies may reach endemic proportions. Gene-editing technology may provide a therapeutic alternative overcoming some of these limitations, though proving its safety and efficacy will most likely require extensive clinical investigation.

2018 - Gene Therapy for Sickle Cell Disease: A Lentiviral Vector Comparison Study [Articolo su rivista]
Urbinati, F.; Campo Fernandez, B.; Masiuk, K. E.; Poletti, V.; Hollis, R. P.; Koziol, C.; Kaufman, M. L.; Brown, D.; Mavilio, F.; Kohn, D. B.

Sickle cell disease (SCD) is an inherited blood disorder caused by a single amino acid substitution in the β-globin chain of hemoglobin. Gene therapy is a promising therapeutic alternative, particularly in patients lacking an allogeneic bone marrow (BM) donor. One of the major challenges for an effective gene therapy approach is the design of an efficient vector that combines high-level and long-Term β-globin expression with high infectivity in primary CD34+ cells. Two lentiviral vectors carrying an anti-sickling β-globin transgene (AS3) were directly compared: The Lenti/βAS3-FB, and Globe-AS3 with and without the FB insulator. The comparison was performed initially in human BM CD34+ cells derived from SCD patients in an in vitro model of erythroid differentiation. Additionally, the comparison was carried out in two in vivo models: First, an NOD SCID gamma mouse model was used to compare transduction efficiency and β-globin expression in human BM CD34+ cells after transplant. Second, a sickle mouse model was used to analyze β-globin expression produced from the vectors tested, as well as hematologic correction of the sickle phenotype. While minor differences were found in the vectors in the in vitro study (2.4-fold higher vector copy number in CD34+ cells when using Globe-AS3), no differences were noted in the overall correction of the SCD phenotype in the in vivo mouse model. This study provides a comprehensive in vitro and in vivo analysis of two globin lentiviral vectors, which is useful for determining the optimal candidate for SCD gene therapy.

2018 - Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human b-globin locus [Articolo su rivista]
Antoniani, C.; Meneghini, V.; Lattanzi, A.; Felix, T.; Romano, O.; Magrin, E.; Weber, L.; Pavani, G.; Hoss, S. E.; Kurita, R.; Nakamura, Y.; Cradick, T. J.; Lundberg, A. S.; Porteus, M.; Amendola, M.; Nemer, W. E.; Cavazzana, M.; Mavilio, F.; Miccio, A.

Naturally occurring, large deletions in the b-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and b-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the d- and b-globin genes and a putative g-d intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in g-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the b-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of g-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of b-hemoglobinopathies.

2018 - Interactions between Retroviruses and the Host Cell Genome [Articolo su rivista]
Poletti, V.; Mavilio, F.

Replication-defective retroviral vectors have been used for more than 25 years as a tool for efficient and stable insertion of therapeutic transgenes in human cells. Patients suffering from severe genetic diseases have been successfully treated by transplantation of autologous hematopoietic stem-progenitor cells (HSPCs) transduced with retroviral vectors, and the first of this class of therapies, Strimvelis, has recently received market authorization in Europe. Some clinical trials, however, resulted in severe adverse events caused by vector-induced proto-oncogene activation, which showed that retroviral vectors may retain a genotoxic potential associated to proviral integration in the human genome. The adverse events sparked a renewed interest in the biology of retroviruses, which led in a few years to a remarkable understanding of the molecular mechanisms underlying retroviral integration site selection within mammalian genomes. This review summarizes the current knowledge on retrovirus-host interactions at the genomic level, and the peculiar mechanisms by which different retroviruses, and their related gene transfer vectors, integrate in, and interact with, the human genome. This knowledge provides the basis for the development of safer and more efficacious retroviral vectors for human gene therapy.

2018 - Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia [Articolo su rivista]
Lidonnici, M. R.; Paleari, Y.; Tiboni, F.; Mandelli, G.; Rossi, C.; Vezzoli, M.; Aprile, A.; Lederer, C. W.; Ambrosi, A.; Chanut, F.; Sanvito, F.; Calabria, A.; Poletti, V.; Mavilio, F.; Montini, E.; Naldini, L.; Cristofori, P.; Ferrari, G.

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for β-thalassemia, we performed in vitro and in vivo studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of β-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for β-thalassemia.

2018 - Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling βAS3 Globin for Gene Therapy for Sickle Cell Disease [Articolo su rivista]
Poletti, Valentina; Urbinati, Fabrizia; Charrier, Sabine; Corre, Guillaume; Hollis, Roger P.; Campo Fernandez, Beatriz; Martin, Samia; Rothe, Michael; Schambach, Axel; Kohn, Donald B.; Mavilio, Fulvio

Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%–80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12–14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3.

2018 - Preclinical Development of a Lentiviral Vector for Gene Therapy of X-Linked Severe Combined Immunodeficiency [Articolo su rivista]
Poletti, V.; Charrier, S.; Corre, G.; Gjata, B.; Vignaud, A.; Zhang, F.; Rothe, M.; Schambach, A.; Gaspar, H. B.; Thrasher, A. J.; Mavilio, F.

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the interleukin-2 receptor γ chain gene (IL2RG), and it is characterized by profound defects in T, B, and natural killer (NK) cell functions. Transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with early murine leukemia retrovirus (MLV)-derived gammaretroviral vectors showed restoration of T cell immunity in patients, but it resulted in vector-induced insertional oncogenesis. We developed a self-inactivating (SIN) lentiviral vector carrying a codon-optimized human IL2RG cDNA driven by the EF1α short promoter (EFS-IL2RG), and we tested its efficacy and safety in vivo by transplanting transduced Il2rg-deficient Lin− HSPCs in an Il2rg−/−/Rag2−/− mouse model. The study showed restoration of T, B, and NK cell counts in bone marrow and peripheral blood and normalization of thymus and spleen cellularity and architecture. High-definition insertion site analysis defined the EFS-IL2RG genomic integration profile, and it showed no sign of vector-induced clonal selection or skewing in primarily and secondarily transplanted animals. The study enables a phase I/II clinical trial aimed at restoring both T and B cell immunity in SCID-X1 children upon non-myeloablative conditioning.

2018 - The Pharmacology of Gene and Cell Therapy [Articolo su rivista]
Conlon, T. J.; Mavilio, F.

2017 - Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System [Articolo su rivista]
Lattanzi, A.; Moiani, A.; Izmiryan, A.; Martin, S.; Mavilio, F.; Bovolenta, M.; Duguez, S.; Mamchaoui, K.; Mouly, V.; Barbon, E.; Bernardi, F.

Exonic duplications account for 10%–15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one guide RNA (gRNA) directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.

2017 - Developing gene and cell therapies for rare diseases: An opportunity for synergy between academia and industry [Articolo su rivista]
Mavilio, F.

For the last 20 years, academic research has been the major, and often only, driving force behind the spectacular development of gene transfer technology for the therapy of rare genetic diseases. Investors and industry became eventually interested in gene and cell therapy, due to the success of a series of pioneering clinical trials that proved efficacy and safety of last-generation technology, and to favorable orphan drug legislation in both Europe and the United States. Developing this forms of therapy is however complex and requires skills and knowledge not necessary available to the industry, which is better placed to develop processes and products and put them on the market. Cooperation between academia and industry is an opportunity to de-risk innovative approaches and ensure a faster and more economical development of therapies for diseases with high unmet medical needs and low-profit expectations.

2017 - Evaluation of tolerance to lentiviral LV-RPE65 gene therapy vector after subretinal delivery in non-human primates [Articolo su rivista]
Matet, A.; Kostic, C.; Bemelmans, A. -P.; Moulin, A.; Rosolen, S. G.; Martin, S.; Mavilio, F.; Amirjanians, V.; Stieger, K.; Lorenz, B.; Behar-Cohen, F.; Arsenijevic, Y.

Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, strategies that have reached the clinical stages rely on adeno-associated viral vectors and two of them documented limited long-term effect. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restored protein expression and cone function in RPE65-deficient mice. In this study, we evaluated the ocular and systemic tolerances of this lentiviral-based therapy (LV-RPE65) on healthy nonhuman primates (NHPs), without adjuvant systemic anti-inflammatory prophylaxis. For the first time, we describe the early kinetics of retinal detachment at 2, 4, and 7 days after subretinal injection using multimodal imaging in 5 NHPs. We revealed prolonged reattachment times in LV-RPE65–injected eyes compared to vehicle-injected eyes. Low- (n = 2) and high-dose (n = 2) LV-RPE65–injected eyes presented a reduction of the outer nuclear and photoreceptor outer segment layer thickness in the macula, that was more pronounced than in vehicle-injected eyes (n = 4). All LV-RPE65–injected eyes showed an initial perivascular reaction that resolved spontaneously within 14 days. Despite foveal structural changes, full-field electroretinography indicated that the overall retinal function was preserved over time and immunohistochemistry identified no difference in glial, microglial, or leucocyte ocular activation between low-dose, high-dose, and vehicle-injected eyes. Moreover, LV-RPE65–injected animals did not show signs of vector shedding or extraocular targeting, confirming the safe ocular restriction of the vector. Our results evidence a limited ocular tolerance to LV-RPE65 after subretinal injection without adjuvant anti-inflammatory prophylaxis, with complications linked to this route of administration necessitating to block this transient inflammatory event.

2017 - Gene Therapy Approaches to Hemoglobinopathies [Capitolo/Saggio]
Ferrari, G.; Cavazzana, M.; Mavilio, F.

Gene therapy for hemoglobinopathies is currently based on transplantation of autologous hematopoietic stem cells genetically modified with a lentiviral vector expressing a globin gene under the control of globin transcriptional regulatory elements. Preclinical and early clinical studies showed the safety and potential efficacy of this therapeutic approach as well as the hurdles still limiting its general application. In addition, for both beta-thalassemia and sickle cell disease, an altered bone marrow microenvironment reduces the efficiency of stem cell harvesting as well as engraftment. These hurdles need be addressed for gene therapy for hemoglobinopathies to become a clinical reality.

2017 - Gene therapy for Wiskott-Aldrich syndrome in a severely affected adult [Articolo su rivista]
Morris, E. C.; Fox, T.; Chakraverty, R.; Tendeiro, R.; Snell, K.; Rivat, C.; Grace, S.; Gilmour, K.; Workman, S.; Buckland, K.; Butler, K.; Chee, R.; Salama, A. D.; Ibrahim, H.; Hara, H.; Duret, C.; Mavilio, F.; Male, F.; Bushman, F. D.; Galy, A.; Burns, S. O.; Gaspar, H. B.; Thrasher, A. J.

Until recently, hematopoietic stem cell transplantation was the only curative option for Wiskott-Aldrich syndrome (WAS). The first attempts at gene therapy for WAS using a ϒ-retroviral vector improved immunological parameters substantially but were complicated by acute leukemia as a result of insertional mutagenesis in a high proportion of patients. More recently, treatment of children with a state-of-the-art self-inactivating lentiviral vector (LV-w1.6 WASp) has resulted in significant clinical benefit without inducing selection of clones harboring integrations near oncogenes. Here, we describe a case of a presplenectomized 30-year-old patient with severe WAS manifesting as cutaneous vasculitis, inflammatory arthropathy, intermittent polyclonal lymphoproliferation, and significant chronic kidney disease and requiring long-term immunosuppressive treatment. Following reduced-intensity conditioning, there was rapid engraftment and expansion of a polyclonal pool of transgene-positive functional T cells and sustained gene marking in myeloid and B-cell lineages up to 20 months of observation. The patient was able to discontinue immunosuppression and exogenous immunoglobulin support, with improvement in vasculitic disease and proin-flammatory markers. Autologous gene therapy using a lentiviral vector is a viable strategy for adult WAS patients with severe chronic disease complications and for whom an allogeneic procedure could present an unacceptable risk. This trial was registered at as #NCT01347242.

2017 - Gene transfer into hematopoietic stem cells reduces HLH manifestations in a murine model of Munc13-4 deficiency [Articolo su rivista]
Soheili, T.; Durand, A.; Sepulveda, F. E.; Riviere, J.; Lagresle-Peyrou, C.; Sadek, H.; De Saint Basile, G.; Martin, S.; Mavilio, F.; Cavazzana, M.; Andre-Schmutz, I.

Patients with mutations in the UNC13D gene (coding for Munc13-4 protein) suffer from familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a life-threatening immune and hyperinflammatory disorder. The only curative treatment is allogeneic hematopoietic stem cell (HSC) transplantation, although the posttreatment survival rate is not satisfactory. Here, we demonstrate the curative potential of UNC13D gene correction of HSCs in a murine model of FHL3. We generated a self-inactivating lentiviral vector, used it to complement HSCs from Unc13d-deficient (Jinx) mice, and transplanted the cells back into the irradiated Jinx recipients. This procedure led to complete reconstitution of the immune system (ie, to wild-type levels). The recipients were then challenged with lymphocytic choriomeningitis virus to induce hemophagocytic lymphohistiocytosis (HLH)-like manifestations. All the clinical and biological signs of HLH were significantly reduced in mice having undergone HSC UNC13D gene correction than in nontreated animals. This beneficial effect was evidenced by the correction of blood cytopenia, body weight gain, normalization of the body temperature, decreased serum interferon-g level, recovery of liver damage, and decreased viral load. These improvements can be explained by the restoration of the CD81 T lymphocytes' cytotoxic function (as demonstrated here in an in vitro degranulation assay). Overall, our results demonstrate the efficacy of HSC gene therapy in an FHL-like setting of immune dysregulation.

2017 - Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy [Articolo su rivista]
Le Guiner, C.; Servais, L.; Montus, M.; Larcher, T.; Fraysse, B.; Moullec, S.; Allais, M.; Francois, V.; Dutilleul, M.; Malerba, A.; Koo, T.; Thibaut, J. -L.; Matot, B.; Devaux, M.; Le Duff, J.; Deschamps, J. -Y.; Barthelemy, I.; Blot, S.; Testault, I.; Wahbi, K.; Ederhy, S.; Martin, S.; Veron, P.; Georger, C.; Athanasopoulos, T.; Masurier, C.; Mingozzi, F.; Carlier, P.; Gjata, B.; Hogrel, J. -Y.; Adjali, O.; Mavilio, F.; Voit, T.; Moullier, P.; Dickson, G.

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients.

2017 - Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions [Articolo su rivista]
Romano, Oriana; Cifola, Ingrid; Poletti, Valentina; Severgnini, Marco; Peano, Clelia; De Bellis, Gianluca; Mavilio, Fulvio; Miccio, Annarita

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs. The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

2017 - Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs [Articolo su rivista]
Mack, D. L.; Poulard, K.; Goddard, M. A.; Latournerie, V.; Snyder, J. M.; Grange, R. W.; Elverman, M. R.; Denard, J.; Veron, P.; Buscara, L.; Le Bec, C.; Hogrel, J. -Y.; Brezovec, A. G.; Meng, H.; Yang, L.; Liu, F.; O'Callaghan, M.; Gopal, N.; Kelly, V. E.; Smith, B. K.; Strande, J. L.; Mavilio, F.; Beggs, A. H.; Mingozzi, F.; Lawlor, M. W.; Buj-Bello, A.; Childers, M. K.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

2016 - Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation [Articolo su rivista]
Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations.

2016 - Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy [Articolo su rivista]
Armbruster, N.; Lattanzi, A.; Jeavons, M.; Van Wittenberghe, L.; Gjata, B.; Marais, T.; Martin, S.; Vignaud, A.; Voit, T.; Mavilio, F.; Barkats, M.; Buj-Bello, A.

Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery.

2016 - Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment [Articolo su rivista]
Romano, Oriana; Peano, Clelia; Tagliazucchi, Guidantonio Malagoli; Petiti, Luca; Poletti, Valentina; Cocchiarella, Fabienne; Rizzi, Ermanno; Severgnini, Marco; Cavazza, Alessia; Rossi, Claudia; Pagliaro, Pasqualepaolo; Ambrosi, Alessandro; Ferrari, Giuliana; Bicciato, Silvio; De Bellis, Gianluca; Mavilio, Fulvio; Miccio, Annarita

Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

2015 - A single epidermal stem cell strategy for safe ex vivo gene therapy [Articolo su rivista]
Droz‐Georget Lathion, Stéphanie; Rochat, Ariane; Knott, Graham; Recchia, Alessandra; Martinet, Danielle; Benmohammed, Sara; Grasset, Nicolas; Zaffalon, Andrea; Besuchet Schmutz, Nathalie; Savioz‐dayer, Emmanuelle; Beckmann, Jacques Samuel; Rougemont, Jacques; Mavilio, Fulvio; Barrandon, Yann

There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self‐inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non‐blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole‐genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long‐term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by‐passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene‐editing technologies like zinc finger nucleases, TALENs and homologous recombination for next‐generation gene therapy.

2015 - Genome-wide definition of promoter and enhancer usage during neural induction of human embryonic stem cells [Articolo su rivista]
Poletti, Valentina; Delli Carri, Alessia; Malagoli Tagliazucchi, Guidantonio; Faedo, Andrea; Petiti, Luca; Mazza, Emilia Maria Cristina; Peano, Clelia; De Bellis, Gianluca; Bicciato, Silvio; Miccio, Annarita; Cattaneo, Elena; Mavilio, Fulvio

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a "bivalent" histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate.

2015 - Nuclear architecture dictates HIV-1 integration site selection [Articolo su rivista]
Marini, Bruna; Kertesz Farkas, Attila; Ali, Hashim; Lucic, Bojana; Lisek, Kamil; Manganaro, Lara; Pongor, Sandor; Luzzati, Roberto; Recchia, Alessandra; Mavilio, Fulvio; Giacca, Mauro; Lusic, Marina

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome1–4. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This regioncontains a seriesof cellular genes,which arepreferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associateddomains5 and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellularNup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome.These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

2015 - Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome [Articolo su rivista]
Hacein-Bey Abina, S.; Gaspar, H. B.; Blondeau, J.; Caccavelli, L.; Charrier, S.; Buckland, K.; Picard, C.; Six, E.; Himoudi, N.; Gilmour, K.; Mcnicol, A. -M.; Hara, H.; Xu-Bayford, J.; Rivat, C.; Touzot, F.; Mavilio, F.; Lim, A.; Treluyer, J. -M.; Heritier, S.; Lefrere, F.; Magalon, J.; Pengue-Koyi, I.; Honnet, G.; Blanche, S.; Sherman, E. A.; Male, F.; Berry, C.; Malani, N.; Bushman, F. D.; Fischer, A.; Thrasher, A. J.; Galy, A.; Cavazzana, M.

IMPORTANCE: Wiskott-Aldrich syndrome is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA antigen-matched allogeneic hematopoietic stem cell (HSC) transplantation is often curative but is associated with significant comorbidity. OBJECTIVE: To assess the outcomes and safety of autologous HSC gene therapy in Wiskott-Aldrich syndrome. DESIGN, SETTING, AND PARTICIPANTS: Gene-corrected autologous HSCs were infused in 7 consecutive patients with severe Wiskott-Aldrich syndrome lacking HLA antigen-matched related or unrelated HSC donors (age range, 0.8-15.5 years; mean, 7 years) following myeloablative conditioning. Patients were enrolled in France and England and treated between December 2010 and January 2014. Follow-up of patients in this intermediate analysis ranged from 9 to 42 months. INTERVENTION: A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector. MAIN OUTCOMES AND MEASURES: Primary outcomes were improvement at 24 months in eczema, frequency and severity of infections, bleeding tendency, and autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and hematological characteristics and evidence of safety through vector integration analysis. RESULTS: Six of the 7 patients were alive at the time of last follow-up (mean and median follow-up, 28 months and 27 months, respectively) and showed sustained clinical benefit. One patient died 7 months after treatment of preexisting drug-resistant herpes virus infection. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5 of 5 patients. No severe bleeding episodes were recorded after treatment, and at last follow-up, all 6 surviving patients were free of blood product support and thrombopoietic agonists. Hospitalization days were reduced from a median of 25 days during the 2 years before treatment to a median of 0 days during the 2 years after treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis. CONCLUSIONS AND RELEVANCE: This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcomes and safety.

2015 - Perspectives on Best Practices for Gene Therapy Programs [Articolo su rivista]
Cheever, Thomas R; Berkley, Dale; Braun, Serge; Brown, Robert H.; Byrne, Barry J.; Chamberlain, Jeffrey S.; Cwik, Valerie; Duan, Dongsheng; Federoff, Howard J.; High, Katherine A.; Kaspar, Brian K.; Klinger, Katherine W.; Larkindale, Jane; Lincecum, John; Mavilio, Fulvio; Mcdonald, Cheryl L.; Mclaughlin, James; Weiss McLeod, Bonnie; Mendell, Jerry R.; Nuckolls, Glen; Stedman, Hansell H.; Tagle, Danilo A.; Vandenberghe, Luk H.; Wang, Hao; Wernett, Pamela J.; Wilson, James M.; Porter, John D.; Gubitz, Amelie K.

With recent successes in gene therapy trials for hemophilia and retinal diseases, the promise and prospects for gene therapy are once again garnering significant attention. To build on this momentum, the National Institute of Neurological Disorders and Stroke and the Muscular Dystrophy Association jointly hosted a workshop in April 2014 on "Best Practices for Gene Therapy Programs," with a focus on neuromuscular disorders. Workshop participants included researchers from academia and industry as well as representatives from the regulatory, legal, and patient advocacy sectors to cover the gamut from preclinical optimization to intellectual property concerns and regulatory approval. The workshop focused on three key issues in the field: (1) establishing adequate scientific premise for clinical trials in gene therapy, (2) addressing regulatory process issues, and (3) intellectual property and commercialization issues as they relate to gene therapy. The outcomes from the discussions at this workshop are intended to provide guidance for researchers and funders in the gene therapy field.

2014 - Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy [Articolo su rivista]
Childers, Martin K.; Joubert, Romain; Poulard, Karine; Moal, Christelle; Grange, Robert W.; Doering, Jonathan A.; Lawlor, Michael W.; Rider, Branden E.; Jamet, Thibaud; Danièle, Nathalie; Martin, Samia; Rivière, Christel; Soker, Thomas; Hammer, Caroline; Van Wittenberghe, Laetitia; Lockard, Mandy; Guan, Xuan; Goddard, Melissa; Mitchell, Erin; Barber, Jane; Williams, J. Koudy; Mack, David L.; Furth, Mark E.; Vignaud, Alban; Masurier, Carole; Mavilio, Fulvio; Moullier, Philippe; Beggs, Alan H.; Buj Bello, Anna

Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients

2014 - Genome-wide analysis of alpharetroviral integration in human hematopoietic stem/progenitor cells [Articolo su rivista]
Moiani, Arianna; Suerth, Julia Debora; Gandolfi, Francesco; Rizzi, Ermanno; Severgnini, Marco; De Bellis, Gianluca; Schambach, Axel; Mavilio, Fulvio

Gene transfer vectors derived from gamma-retroviruses or lentiviruses are currently used for the gene therapy of genetic or acquired diseases. Retroviral vectors display a non-random integration pattern in the human genome, targeting either regulatory regions (gamma-retroviruses) or the transcribed portion of expressed genes (lentiviruses), and have the potential to deregulate gene expression at the transcriptional or post-transcriptional level. A recently developed alternative vector system derives from the avian sarcoma-leukosis alpha-retrovirus (ASLV) and shows favorable safety features compared to both gamma-retroviral and lentiviral vectors in preclinical models. We performed a high-throughput analysis of the integration pattern of self-inactivating (SIN) alpha-retroviral vectors in human CD34+ hematopoietic stem/progenitor cells (HSPCs) and compared it to previously reported gamma-retroviral and lentiviral vectors integration profiles obtained in the same experimental setting. Compared to gamma-retroviral and lentiviral vectors, the SIN-ASLV vector maintains a preference for open chromatin regions, but shows no bias for transcriptional regulatory elements or transcription units, as defined by genomic annotations and epigenetic markers (H3K4me1 and H3K4me3 histone modifications). Importantly, SIN-ASLV integrations do not cluster in hot spots and target potentially dangerous genomic loci, such as the EVI2A/B, RUNX1 and LMO2 proto-oncogenes at a virtually random frequency. These characteristics predict a safer profile for ASLV-derived vectors for clinical applications

2014 - Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells [Articolo su rivista]
Turchiano, Giandomenico; Latella, Maria Carmela; Gogol Doring, Andreas; Cattoglio, Claudia; Mavilio, Fulvio; Izsvak, Zsuzsanna; Ivics, Zoltan; Recchia, Alessandra

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity ‘‘sandwich’’ (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications

2014 - Repairing without cutting: A safer alternative to gene correction? [Articolo su rivista]
Mavilio, Fulvio


2013 - Collagen VII gene delivery via Sleeping Beauty transposon in COL7A1-deficient keratinocytes from epidermolysis bullosa patients [Abstract in Atti di Convegno]
Latella, Maria Carmela; Cocchiarella, Fabienne; Turchiano, Giandomenico; Gonçalves, Manuel; Larcher, Fernando; Mavilio, Fulvio; Izsvak, Zsuzsanna; Ivics, Zoltan; Recchia, Alessandra

Autosomal recessive epidermolysis bullosa (RDEB) is a genetic skin adhesion defect caused by mutations in the type VII collagen gene (COL7A1). Although full-length type-VII collagen can be successfully produced in human keratinocytes following retroviral vector transduction, genetic instability due to the large size (9kb) and highly repetitive nature of the gene sequence remains problematic. The Sleeping Beauty (SB) transposon-based integration system can potentially overcome these issues by taking advantage of the hyperactive SB100X transposase in combination with the pT2 transposon..

2013 - Deletion of the LTR Enhancer/Promoter Has No Impact on the Integration Profile of MLV Vectors in Human Hematopoietic Progenitors [Articolo su rivista]
Moiani, A.; Miccio, A.; Rizzi, E.; Severgnini, M.; Pellin, D.; Suerth, J. D.; Baum, C.; de Bellis, G.; Mavilio, F.

Moloney murine leukemia virus (MLV)-derived gamma-retroviral vectors integrate preferentially near transcriptional regulatory regions in the human genome, and are associated with a significant risk of insertional gene deregulation. Self-inactivating (SIN) vectors carry a deletion of the U3 enhancer and promoter in the long terminal repeat (LTR), and show reduced genotoxicity in pre-clinical assays. We report a high-definition analysis of the integration preferences of a SIN MLV vector compared to a wild-type-LTR MLV vector in the genome of CD34(+) human hematopoietic stem/progenitor cells (HSPCs). We sequenced 13,011 unique SIN-MLV integration sites and compared them to 32,574 previously generated MLV sites in human HSPCs. The SIN-MLV vector recapitulates the integration pattern observed for MLV, with the characteristic clustering of integrations around enhancer and promoter regions associated to H3K4me3 and H3K4me1 histone modifications, specialized chromatin configurations (presence of the H2A.Z histone variant) and binding of RNA Pol II. SIN-MLV and MLV integration clusters and hot spots overlap in most cases and are generated at a comparable frequency, indicating that the reduced genotoxicity of SIN-MLV vectors in hematopoietic cells is not due to a modified integration profile.

2013 - Genotoxic signature in cord blood cells of newborns exposed in utero to a zidovudine-based antiretroviral combination [Articolo su rivista]
Isabelle Andre, Schmutz; Liliane Dal, Cortivo; Emmanuelle, Six; Sophie, Kaltenbach; Fabienne, Cocchiarella; Jerome Le, Chenadec; Nicolas, Cagnard; Anne Gael, Cordier; Alexandra, Benachi; Laurent, Mandelbrot; Elie, Azria; Naima, Bouallag; Sonia, Luce; Brigitte, Ternaux; Christian, Reimann; Patrick, Revy; Isabelle Radford, Weiss; Cristina, Leschi; Recchia, Alessandra; Mavilio, Fulvio; Marina Cavazzana, Calvo*; Stéphane, Blanche

Background: Zidovudine experimental genotoxicity has been established. Objective of the study was to identify genotoxicity markers in cord blood cells from newborns exposed in utero to ARV combinations containing zidovudine. Methods: Cells were investigated by karyotyping and gene expression analysis of the CD34+ hematopoietic stem/progenitor cell compartment (HPC). Results: Karyotyping of the cord blood cells from 15 ARV-exposed newborns and 12 controls revealed a higher proportion of aneuploid cells in the exposed group (median [IQR]: 18.8% [10.0-26.7] vs. 6.6 % [3.1-11.7], p<0.001). All chromosomes were involved with a random distribution of these alterations. Gene expression profiling of CD34+ HPCs from seven ARV-exposed and six control newborns revealed >300 genes significantly up- or down-regulated (p<0.05) by at least 1.5-fold in the exposed group. Significant alterations of genes involved in the control of cell cycle, mitotic check-points and DNA repair were identified. Although this study does not allow discrimination between the roles of each of the three drugs, both cytogenetic and transcriptional findings are similar to those in cellular experiments using zidovudine alone. Conclusion: The cord blood cells —including hematopoietic stem cells— from newborns exposed in utero to a zidovudine-based ARV combination present cytogenetic and transcriptional abnormalities compatible with DNA damage.

2013 - IL-7 and IL-15 instruct the generation of human memory stem T cells from naïve precursors [Articolo su rivista]
N., Cieri; B., Camisa; Cocchiarella, Fabienne; Forcato, Mattia; G., Oliveira; E., Provasi; A., Bondanza; C., Bordignon; J., Peccatori; F., Ciceri; M. T., Lupo Stanghellini; Mavilio, Fulvio; A., Mondino; Bicciato, Silvio; Recchia, Alessandra; C., Bonini

Long-living memory stem T cells (TSCM) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here we show that it is possible to differentiate in vitro, expand and gene modify in clinically compliant conditions CD8+ TSCM lymphocytes starting from naïve precursors. Requirements for the generation of this T-cell subset, described as CD62L+CCR7+CD45RA+CD45R0+IL- 7Rα+CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly TSCM accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T lymphocyte subset, intermediate between naïve and central memory cells. When transplanted in immunodeficient mice, gene-modified naïve-derived TSCM prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GvHD. Furthermore, gene-modified TSCM are the only T-cell subset able to expand and mediate GvHD upon serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for TSCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy

2013 - Mechanisms of retroviral integration and mutagenesis [Articolo su rivista]
Cavazza, Alessia; Moiani, Arianna; Mavilio, Fulvio

Gene transfer vectors derived from oncoretroviruses or lentiviruses are the most robust and reliable tools to stably integrate therapeutic transgenes in human cells for clinical applications. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. In addressing these issues, the gene therapy community offered a spectacular example of how scientific knowledge and technology can be put to work to understand the causes of unpredicted side effects, design new vectors, and develop tools and models to predict their safety and efficacy. As an added benefit, these efforts brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. This review summarizes the current knowledge on the interactions between retroviruses and the human genome and addresses the impact of target site selection on the safety of retroviral vector-mediated gene therapy

2013 - Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication [Articolo su rivista]
Di Nunzio, F.; Fricke, T.; Miccio, A.; Valle-Casuso, J. C.; Perez, P.; Souque, P.; Rizzi, E.; Severgnini, M.; Mavilio, F.; Charneau, P.; Diaz-Griffero, F.

The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration.

2013 - RD2-molpack-chim3, a packaging cell line for stable production of lentiviral vectors for Anti-HIV gene therapy [Articolo su rivista]
Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

2013 - Self-inactivating MLV vectors have a reduced genotoxic profile in human epidermal keratinocytes [Articolo su rivista]
Cavazza, A.; Cocchiarella, F.; Bartholomae, C.; Schmidt, M.; Pincelli, C.; Larcher, F.; Mavilio, F.

Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN-HIV vector showed the expected preference for transcribed genes while the SIN-MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN-MLV design has the lowest activity compared to both MLV and SIN-HIV vectors. This study indicates that SIN-MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders. © 2013 Macmillan Publishers Limited All rights reserved.

2013 - Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination [Articolo su rivista]
Coluccio, Andrea; Miselli, Francesca; Angelo, Lombardo; Marconi, Alessandra; Guidantonio Malagoli, Tagliazucchi; Manuel A., Gonçalves; Pincelli, Carlo; Giulietta, Maruggi; Marcela Del, Rio; Luigi, Naldini; Fernando, Larcher; Mavilio, Fulvio; Recchia, Alessandra

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a “safe harbor” locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of >20% in a human keratinocyte cell line, >10% in immortalized keratinocytes, and <1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

2013 - Translating the genomics revolution: The need for an international gene therapy consortium for monogenic diseases [Articolo su rivista]
Tremblay, Jacques P; Xiao, Xiao; Aartsma Rus, Annemieke; Barbas, Carlos; Blau, Helen M.; Bogdanove, Adam J.; Boycott, Kym; Braun, Serge; Breakefield, Xandra O.; Bueren, Juan A.; Buschmann, Michael; Byrne, Barry J.; Calos, Michele; Cathomen, Toni; Chamberlain, Jeffrey; Chuah, Marinee; Cornetta, Kenneth; Davies, Kay E.; Dickson, J. George; Duchateau, Philippe; Flotte, Terence R.; Gaudet, Daniel; Gersbach, Charles A.; Gilbert, Renald; Glorioso, Joseph; Herzog, Roland W.; High, Katherine A.; Huang, Wenlin; Huard, Johnny; Joung, J. Keith; Liu, Depei; Liu, Dexi; Lochmüller, Hanns; Lustig, Lawrence; Martens, Jeffrey; Massie, Bernard; Mavilio, Fulvio; Mendell, Jerry R.; Nathwani, Amit; Ponder, Katherine; Porteus, Matthew; Puymirat, Jack; Samulski, Jude; Takeda, Shin'Ichi; Thrasher, Adrian; Vandendriessche, Thierry; Wei, Yuquan; Wilson, James M.; Wilton, Steve D.; Wolfe, John H.; Gao, Guangping


2012 - Alternative splicing caused by lentiviral integration in the human genome [Capitolo/Saggio]
Moiani, A.; Mavilio, F.

Gene transfer vectors derived from murine oncoretroviruses or human lentiviruses are widely used in human gene therapy. Integration of these vectors in the human genome may, however, have genotoxic effects, caused by deregulation of gene expression at the transcriptional or posttranscriptional level. In particular, integration of lentiviral vectors within transcribed genes has a significant potential to affect their expression by interfering with splicing and polyadenylation of primary transcripts. Aberrant splicing is caused by the usage of both constitutive and cryptic splice sites located in the retroviral backbone as well as in the gene expression cassettes. We describe a set of simple methods that allow the identification of chimeric transcripts generated by the insertion of a lentiviral vector within genes and the evaluation of their relative abundance. Identification of the splice sites, either constitutive or cryptic, that are frequently used by the cell splicing machinery within a given vector provides a useful resource to attempt recoding of the vector with the objective of reducing its potential genotoxicity in a clinical context. © 2012 Elsevier Inc. All rights reserved.

2012 - Gene therapies need new development models [Articolo su rivista]
Mavilio, Fulvio


2012 - Gene therapy of skin adhesion disorders (mini review) [Articolo su rivista]
Cavazza, Alessia; Mavilio, Fulvio

Gene therapy is a potential treatment for severe inherited disorders for which there is little hope of finding a conventional cure. These include lethal diseases like immunodeficiencies and metabolic disorders, and non lethal conditions associated to poor quality of life and life-long symptomatic treatments, like muscular dystrophy, cystic fibrosis or thalassemia. Skin adhesion defects belong to both groups. For the non-lethal forms, gene therapy, or transplantation of cultured skin derived from genetically corrected epidermal stem cells, represents a very attractive therapeutic option, and potentially a definitive treatment. Recent advances in gene transfer and stem cell culture technology are making this option closer than ever. This paper critically reviews the progress and prospects of gene therapy for skin adhesion defects, and the factors currently limiting its development.

2012 - Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts [Articolo su rivista]
Moiani, Arianna; Paleari, Ylenia; Sartori, Daniela; Mezzadra, Riccardo; Miccio, Annarita; Cattoglio, Claudia; Cocchiarella, Fabienne; Lidonnici, Maria Rosa; Ferrari, Giuliana; Mavilio, Fulvio

Retroviral vectors integrate in genes and regulatory elements and may cause transcriptional deregulation of gene expression in target cells. Integration into transcribed genes also has the potential to deregulate gene expression at the posttranscriptional level by interfering with splicing and polyadenylation of primary transcripts. To examine the impact of retroviral vector integration on transcript splicing, we transduced primary human cells or cultured cells with HIV-derived vectors carrying a reporter gene or a human β-globin gene under the control of a reduced-size locus-control region (LCR). Cells were randomly cloned and integration sites were determined in individual clones. We identified aberrantly spliced, chimeric transcripts in more than half of the targeted genes in all cell types. Chimeric transcripts were generated through the use of constitutive and cryptic splice sites in the HIV 5ι long terminal repeat and gag gene as well as in the β-globin gene and LCR. Compared with constitutively spliced transcripts, most aberrant transcripts accumulated at a low level, at least in part as a consequence of nonsense-mediated mRNA degradation. A limited set of cryptic splice sites caused the majority of aberrant splicing events, providing a strategy for recoding lentiviral vector backbones and transgenes to reduce their potential posttranscriptional genotoxicity.

2012 - Non viral gene transfer via Sleeping beauty transposon for Collagen VII delivery in human primary keratinocytes. [Poster]
Latella, Maria Carmela; Turchiano, Giandomenico; Cocchiarella, Fabienne; Izsvak, Zsuzsanna; Ivics, Zoltan; Mavilio, Fulvio; Recchia, Alessandra

Autosomal recessive epidermolysis bullosa (RDEB) is a genetic skin adhesion defect caused by mutations in the type VII collagen gene (COL7A1). Although full-length type-VII collagen is successfully produced in human keratinocytes by retroviral vectors, genetic instability due to the large size (9kb) and the highly repeated nature of the gene sequence is a persistent problem. The Sleeping-Beauty (SB) transposon-based integration system can potentially overcome these issues by taking advantage of the hyperactive SB100X transposase in combination with the wild-type (pT2) transposon or the “sandwich” version (pSA) that showed robust transposition efficiency in human cells. We molecularly characterized the “sandwich” SB-mediated integrants in epithelial cell lines and in primary keratinocytes. Co-transfecting the transposase together with 10kb-transposon (pT2 or pSA) we observed up to 37% of transposition in HaCaT and in GABEB (generalized atrophic benign epidermolysis bullosa keratinocytes) cells with both transposons. Clonal analysis demonstrated that the transposition events occur with a minimal risk of rearrangements (<3%). LM-PCR based bi-directional sequencing of the transposon-genome junctions shows genuine “cut and paste” activity of the SB hyperactive transposase.

2012 - Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells [Articolo su rivista]
Warrick, Emilie; Garcia, Marta; Chagnoleau, Corinne; Chevallier, Odile; Bergoglio, Valérie; Sartori, Daniela; Mavilio, Fulvio; Angulo, Jaime F.; Avril, Marie Françoise; Sarasin, Alain; Larcher, Fernando; Del Rio, Marcela; Bernerd, Françoise; Magnaldo, Thierry

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (>10(40) cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients.

2012 - Targeted gene integration in human epidermal stem cells by Zinc-finger nuclease-mediated homologous recombination. [Abstract in Atti di Convegno]
Coluccio, Andrea; Lombardo, Angelo; Miselli, Francesca; Holmes, Michael; Gregory, Philip D.; Naldini, Luigi; Recchia, Alessandra; Mavilio, Fulvio

Transplantation of autologous, genetically corrected epidermal stem cells (EpSC) is a potential treatment for junctional epidermolysis bullosa, a genetic skin adhesion disorder. Targeted transgene integration overcomes the issue of random insertional mutagenesis associated with retroviral vectors, and may thus provides a safer gene transfer alternative. We developed a gene-targeting platform based on the use of zinc-finger nucleases (ZFNs) and integrase-defective lentiviral vectors (IDLVs) to insert a transgene by homologous recombination (HR) into the AAVS1 locus on chromosome 19. We evaluated the targeting efficiency in a keratinocyte cell line (HaCaT) by IDLV-mediated delivery of an AAVS1-specific ZFN pair together with an HR construct driving the insertion of a GFP expression cassette into the site of cleavage. We achieved up to 25% of targeted insertion of single copies or concatamers of the GFP cassette into the AAVS1 locus, as analyzed by PCR, Southern blotting and sequencing on individual HaCaT cell clones. Evidence of HR-mediated targeted integration was also obtained at a lower but significant frequency in human primary keratinocyte cultures, by using ZFNs-expressing Adeno vector together with IDLV carrying the GFP expression cassette flanked by AAVS1 homology arms. We observed up to 9% disruption of the ZFN target site, repaired by non-homologous end joining, as evaluated by Cel-1 assay and pyrosequencing. These data suggest that the major limitation of ZFN-mediated targeted integration is represented by poor induction of the HR-dependent DNA repair pathway. To study gene targeting in repopulating keratinocyte stem cells, human skin equivalents derived from keratinocytes co-infected with IDLV donor and AdZFNs vectors were grafted onto immunodeficient (nu/nu) mice. GFP-positive spots were observed for at least 10 weeks in the grafted tissue, confirming that stable integration occurred in transplantable keratinocyte stem cells.

2011 - Correction of murine SCID-X1 by lentiviral gene therapy using a codon-optimized IL2RG gene and minimal pretransplant conditioning [Articolo su rivista]
Huston, Marshall W.; Van Til, Niek P.; Visser, Trudi P.; Arshad, Shazia; Brugman, Martijn H.; Cattoglio, Claudia; Nowrouzi, Ali; Li, Yuedan; Schambach, Axel; Schmidt, Manfred; Baum, Christopher; Von Kalle, Christof; Mavilio, Fulvio; Zhang, Fang; Blundell, Mike P.; Thrasher, Adrian J.; Verstegen, Monique M. A.; Wagemaker, Gerard

Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg(-/-) mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required.

2011 - Defining the lentiviral integrome in human hematopoietic cells [Abstract in Atti di Convegno]
Recchia, Alessandra; Cattoglio, Claudia; Pellin, Danilo; Rizzi, Ermanno; Corti, Giorgio; Di Serio, Clelia; Malani, Nirav; Bushman, Frederick; De Bellis, Gianluca; Mavilio, Fulvio

Retroviral integration is a non-random process, whereby pre-integration complexes of different viruses recognize components or features of the host cell chromatin in a specific fashion. By using deep sequencing technology, we mapped >60,000 MLV and HIV integration sites in the genome of human CD34+ hematopoietic stem/progenitor cells and >16,000 sites in peripheral blood T-lymphocytes, and defined genome-wide integration maps in both cell types. MLV integrations cluster around regulatory elements (promoters, enhancers, and evolutionarily conserved non-coding regions) of genes involved in hematopoietic functions, and to chromatin regions bearing epigenetic marks of active or poised transcription. On the contrary, HIV integrations are clustered in regions marked by histone modifications associated to the body of transcribed genes (H3K36me3 and H2BK5me1), and are under-represented in regulatory regions. Although >90% of the genes targeted by HIV integration are expressed by Affymetrix analysis, expressed genes are not equally targeted. By a rigorous statistical analysis, we define a set of <400 genes that are targeted by HIV at significantly higher frequency than matched random controls after normalization for gene length, and a smaller set of genes that are targeted at significantly lower frequency. Functional clustering analysis shows that highly targeted genes are involved in chromatin remodeling and transcription, and are enriched in housekeeping functions. This analysis identifies a set of “high-risk” genes in hematopoietic cells, the function of which is more likely to be influenced by lentiviral vector integration in clinical gene therapy. Many of these genes are over-represented in collections of lentiviral vector integrations from patients treated by gene therapy, indicating that lentiviral “common integration sites” are determined by the HIV target site selection rather than clonal dominance in vivo.

2011 - Development of a Sleeping Beauty transposon-based integration system for gene transfer in human epithelial cells [Abstract in Atti di Convegno]
Turchiano, Giandomenico; Latella, Maria Carmela; Izsvak, Zsuzsanna; Ivics, Zoltan; Mavilio, Fulvio; Recchia, Alessandra

Transplantation of autologous, genetically corrected epidermal stem cells (EpSC) was successfully used to treat junctional epidermolysis bullosa (EB), a genetic skin adhesion disorder. The dystrophic forms of EB is caused by mutations in the type-VII collagen gene (COL7A1) Delivering the >9 kb COL7A1 cDNA by a retroviral vector is problematic, due to the large size and highly repeated nature of its sequence, which induce genetic rearrangements during reverse transcription and integration. We tested the feasibility of using a non-viral vector system based on Sleeping Beauty (SB)-derived transposons, taking advantages of the recently developed, high-capacity “sandwich” version of the SB transposon and the “hyperactive” SB 100X transposase, which showed high transposition efficiency in human stem cells. We tested the system in HeLa cells and in a keratinocyte cell line (HaCaT), which were co-transfected with the SB 100X transposase and either the normal or the sandwich version of the SB transposon containing a small-size reporter gene (Venus or GFP) expression cassette. In both cell lines, transposition was obtained in up to 80% of the transfected cells with the sandwich transposon, compared to ~50% obtained with the older version. Transposed HaCaT cells were cloned and analysed for integration events. Individual clones carried several copies of the integrated transposon of the predicted size. High-throughput sequencing is under way to analyze the sandwich SB transposon integration characteristics. We then tested a transposon carrying an 8.8-kb cassette, which again showed up to 80% transposition efficiency in transfected cells. Finally, we generated sandwich transposons containing an expression cassette for the COL7A1 cDNA under the control of a constitutive (PGK) or a keratinocyte-specific (K14) promoter, which are currently being tested for integration in HaCaT cells. The SB-based gene delivery system will finally be tested in human primary keratinocyte cultures.

2011 - Estimated comparative integration hotspots identify different behaviors of retroviral gene transfer vectors [Articolo su rivista]
Ambrosi, Alessandro; Glad, Ingrid K.; Pellin, Danilo; Cattoglio, Claudia; Mavilio, Fulvio; Di Serio, Clelia; Frigessi, Arnoldo

Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas.

2011 - Insertion sites in engrafted cells cluster within a limited repertoire of genomic areas after gammaretroviral vector gene therapy [Articolo su rivista]
Deichmann, Annette; Brugman, Martijn H.; Bartholomae, Cynthia C.; Schwarzwaelder, Kerstin; Verstegen, Monique M. A.; Howe, Steven J.; Arens, Anne; Ott, Marion G.; Hoelzer, Dieter; Seger, Reinhard; Grez, Manuel; Hacein Bey Abina, Salima; Cavazzana Calvo, Marina; Fischer, Alain; Paruzynski, Anna; Gabriel, Richard; Glimm, Hanno; Abel, Ulrich; Cattoglio, Claudia; Mavilio, Fulvio; Cassani, Barbara; Aiuti, Alessandro; Dunbar, Cynthia E.; Baum, Christopher; Gaspar, H. Bobby; Thrasher, Adrian J.; Von Kalle, Christof; Schmidt, Manfred; Wagemaker, Gerard

Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.

2011 - Risk assessment in skin gene therapy: Viral-cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors [Articolo su rivista]
Almarza, D.; Bussadori, G.; Navarro, M.; Mavilio, F.; Larcher, F.; Murillas, R.

Cutaneous gene therapy can be envisioned through the use of keratinocyte stem cell clones in which retroviral genotoxic risks can be pre-assessed. While transactivation of cellular genes by the retroviral long terminal repeat enhancer has been proven in experimental and clinical settings, the formation of chimeric viral-cellular transcripts originated by the inefficient termination (read-through) of retroviral transcripts remains to be studied in depth. We now demonstrate the widespread presence of viral-cellular fusion transcripts derived from integrated proviruses in keratinocytes transduced with self-inactivating (SIN) retroviral vectors. We have detected high molecular weight RNAs in northern blot analysis of retroviral vector expression in individual cell clones. Characterization of some of these transcripts revealed that they originate from genes located at the proviral integration sites. One class of transcripts corresponds to fusions of the viral vectors with intronic sequences, terminating at cryptic polyadenylation sites located in introns. A second class comprises fusion transcripts with coding sequences of genes at the integration sites. These are generated through splicing from a cryptic, not previously described donor site in the lentiviral vectors to exons of cellular genes, and have the potential to encode unintended open reading frames, although they are downregulated by cellular mechanisms. Our data contribute to a better understanding of the impact of SIN lentiviral vector integration on cellular gene transcription, and will be helpful in improving the design of this type of vectors. © 2011 Macmillan Publishers Limited All rights reserved.

2011 - Site-specific integration by the adeno-associated virus rep protein [Articolo su rivista]
Recchia, Alessandra; Mavilio, Fulvio

Inserting genetic information at precise locations into the human genome has been the goal of gene transfertechnology for almost two decades. The spectacular progress of mammalian genetics in the last two decades has led to thedevelopment of technology for genome editing and homologous recombination in human somatic cells that is finally approachingefficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locationsby enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could verywell be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adeno-associated virus (AAV), to insert genes at the natural AAV integration site on human chromosome19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesis

2011 - The GATA1-HS2 enhancer allows persistent and position-independent expression of a β-globin transgene [Articolo su rivista]
Miccio, A.; Poletti, V.; Tiboni, F.; Rossi, C.; Antonelli, A.; Mavilio, F.; Ferrari, G.

Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing. © 2011 Miccio et al.

2010 - Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients [Articolo su rivista]
E. A., Roselli; R., Mezzadra; M. C., Frittoli; Maruggi, Giulietta; E., Biral; Mavilio, Fulvio; F., Mastropietro; A., Amato; G., Tonon; C., Refaldi; M. D., Cappellini; M., Andreani; G., Lucarelli; M. G., Roncarolo; S., Marktel; G., Ferrari

Beta-thalassemia is a common monogenic disorder due to mutations in the beta-globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine beta-thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human beta-globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM-derived CD34(+) cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer-related genes. Overall, these results provide a solid rationale for a future clinical translation.

2010 - Gene therapy: back on track? [Articolo su rivista]
Mavilio, Fulvio

Not available

2010 - High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion. [Articolo su rivista]
C., Cattoglio C; G., Maruggi; C., Bartholomae; N., Malani; D., Pellin; Cocchiarella, Fabienne; Z., Magnani; F., Ciceri; A., Ambrosi; C., von Kalle; Bushman, F. D.; C., Bonini; M., Schmidt; Mavilio, Fulvio; Recchia, Alessandra

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

2010 - High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors [Articolo su rivista]
C., Cattoglio; D., Pellin; E., Rizzi; Maruggi, Giulietta; G., Corti; Miselli, Francesca; D., Sartori; A., Guffanti; C., Di Serio; A., Ambrosi; G., De Bellis; Mavilio, Fulvio

Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of &gt; 60 000 integration sites of Moloney murine leukemia virus (MLV)- and HIV-based retroviral vectors in the genome of human CD34(+) multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions

2010 - Integration site selection by retroviruses and retroviral vectors [Capitolo/Saggio]
Cattoglio, C.; Mavilio, F.

2009 - Comprehensive genomic access to vector integration in clinical gene therapy. [Articolo su rivista]
Gabriel, R; Eckenberg, R; Paruzynski, A; Bartholomae, Cc; Nowrouzi, A; Arens, A; Howe, Sj; Recchia, Alessandra; Cattoglio, C; Wang, W; Faber, K; Schwarzwaelder, K; Kirsten, R; Deichmann, A; Ball, Cr; Balaggan, Ks; Yáñez Muñoz, Rj; Ali, Rr; Gaspar, Hb; Biasco, L; Aiuti, A; Cesana, D; Montini, E; Naldini, L; Cohen Haguenauer, O; Mavilio, Fulvio; Thrasher, Aj; Glimm, H; von Kalle, C; Saurin, W; Schmidt, M.

Retroviral vectors have induced subtle clonal skewing inmany gene therapy patients and severe clonal proliferationand leukemia in some of them, emphasizing the needfor comprehensive integration site analyses to assess thebiosafety and genomic pharmacokinetics of vectors andclonal fate of gene-modified cells in vivo. Integration siteanalyses such as linear amplification–mediated PCR(LAM-PCR) require a restriction digest generating unevenlysmall fragments of the genome. Here we show that eachrestriction motif allows for identification of only a fractionof all genomic integrants, hampering the understanding andprediction of biological consequences after vector insertion.We developed a model to define genomic access to theviral integration site that provides optimal restriction motifcombinations and minimizes the percentage of nonaccessibleinsertion loci. We introduce a new nonrestrictive LAM-PCRapproach that has superior capabilities for comprehensiveunbiased integration site retrieval in preclinical andclinical samples independent of restriction motifs andamplification inefficiency

2009 - Epithelial stem cells in corneal regeneration and epidermal gene therapy. [5YIF: 6.94; Citations: 68] [Articolo su rivista]
Pellegrini, Graziella; P., Rama; Mavilio, Fulvio; DE LUCA, Michele

Regenerative medicine refers to innovative therapies aimed at the permanent restoration of diseased tissues and organs. Regeneration of self-renewing tissues requires specific adult stem cells, which need to be genetically modified to correct inherited genetic diseases. Cultures of epithelial stem cells permanently restore severe skin and mucosal defects, and genetically corrected epidermal stem cells regenerate a normal epidermis in patients carrying junctional epidermolysis bullosa. The keratinocyte stem cell is therefore the only cultured stem cell used both in cell therapy and gene therapy clinical protocols. Epithelial stem cell identification, fate and molecular phenotype have been extensively reviewed, but not in relation to tissue regeneration. In this paper we focus on the localization and molecular characterization of human limbal stem cells in relation to corneal regeneration, and the gene therapy of genetic skin diseases by means of genetically modified epidermal stem cells.

2009 - Gene therapy of inherited skin adhesion disorders: a critical overview [Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella; Mavilio, Fulvio

Gene therapy has the potential to treat devastating inherited diseases for which there is little hope of finding a conventional cure. These include lethal diseases, like immunodeficiencies or several metabolic disorders, or conditions associated with a relatively long life expectancy but poor quality of life and expensive and life-long symptomatic treatments, such as muscular dystrophy, cystic fibrosis and thalassaemia. Skin adhesion defects belong to both groups. For the nonlethal forms, gene therapy, or transplantation of cultured skin derived from genetically corrected epidermal stem cells, represents a very attractive therapeutic option, and potentially a definitive treatment. Recent advances in gene transfer and stem cell culture technology are making this option closer than ever. This paper critically reviews the progress and prospects of gene therapy for epidermolysis bullosa, and the technical and nontechnical factors currently limiting its development.

2009 - Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy [Articolo su rivista]
B., Cassani; E., Montini; Maruggi, Giulietta; A., Ambrosi; M., Mirolo; S., Selleri; E., Biral; I., Frugnoli; V., Hernandez Trujillo; C., Di Serio; M. G., Roncarolo; L., Naldini; Mavilio, Fulvio; A., Aiuti

Gene transfer into hematopoietic stem cells by gamma-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34(+) cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vbeta repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at as #NCT00598481 and #NCT00599781.

2009 - PPARδ is a ligand-dependent negative regulator of Vitamin D3-induced monocyte differentiation [Articolo su rivista]
A., Lymboussaki; Gemelli, Claudia; Testa, Anna; Facchini, Giulia; Ferrari, Francesco; Mavilio, Fulvio; Grande, Alexis

A number of reports indicate that peroxisome proliferator-activated receptor (PPAR) delta is involved in the molecular control of monocyte-macrophage differentiation. In this regard, the recent demonstration that PPARdelta is a primary response gene of 1alpha,25-dihydroxyvitamin D3 (VD), i.e. a powerful inducer of such process, allowed us to hypothesize the existence of a cross talk between PPARdelta and VD receptor pathways. To address this issue, we analyzed the effects promoted by stimulation with PPARdelta ligands and by overexpression of this nuclear receptor in monoblastic cell lines undergoing exposure to VD. The results obtained evidenced that, although promoting a weak differentiation effect by themselves, PPARdelta ligands efficiently co-operated with VD treatment. In spite of this, PPARdelta overexpression exerted a remarkable inhibitory effect on monocyte-macrophage differentiation induced by VD that was, at least partly, reverted by stimulation with a highly specific PPARdelta ligand. These data indicate that, although acting through a ligand-dependent modality, PPARdelta is a negative regulator of VD-mediated monocyte differentiation, allowing us to hypothesize a role of the investigated nuclear receptor in the differentiation block of M5 type (monoblastic) acute myeloid leukemias (AMLs). Bioinformatic analysis of a microarray database, containing the expression profiles of 285 AML cases, further supported this hypothesis demonstrating the existence of a subset of M5 type (monoblastic) AMLs that overexpress PPARdelta gene.

2009 - Tracking gene-modified T cells in vivo. [Capitolo/Saggio]
Recchia, Alessandra; Mavilio, Fulvio

Identification, monitoring, and analysis of genetically modified cells in the peripheral blood are an important component of the clinical follow-up of patients treated by hematopoietic cell gene therapy. Analysis of gene-marked peripheral blood cells provides crucial information on gene transfer efficiency as well as on the nature and characteristics of the genetically modified cells, and may provide early evidence of the occurrence of potentially detrimental side effects. T lymphocytes are a convenient target for this type of analysis, due to their abundance and their relatively long life span in vivo. Tracking of gene-marked T cells is based on relatively simple, FACS- and PCR-based techniques, which may be applied to monitoring genetically modified T cells as well as T cells derived from transplanted, genetically modified hematopoietic stem cells. This chapter provides a description of these techniques and clues to their rational use in a clinical setting.

2009 - Transcription factor binding sites are genetic determinants of retroviral integration in the human genome. [Articolo su rivista]
Felice, B; Cattoglio, C; Cittaro, D; Testa, Anna; Miccio, Annarita; Ferrari, G; Luzi, L; Recchia, Alessandra; Mavilio, Fulvio

Gamma-retroviruses and lentiviruses integrate non-randomly in mammalian genomes, with specific preferences for active chromatin, promoters and regulatory regions. Gene transfer vectors derived from gamma-retroviruses target at high frequency genes involved in the control of growth, development and differentiation of the target cell, and may induce insertional tumors or pre-neoplastic clonal expansions in patients treated by gene therapy. The gene expression program of the target cell is apparently instrumental in directing gamma-retroviral integration, although the molecular basis of this phenomenon is poorly understood. We report a bioinformatic analysis of the distribution of transcription factor binding sites (TFBSs) flanking >4,000 integrated proviruses in human hematopoietic and non-hematopoietic cells. We show that gamma-retroviral, but not lentiviral vectors, integrate in genomic regions enriched in cell-type specific subsets of TFBSs, independently from their relative position with respect to genes and transcription start sites. Analysis of sequences flanking the integration sites of Moloney leukemia virus (MLV)- and human immunodeficiency virus (HIV)-derived vectors carrying mutations in their long terminal repeats (LTRs), and of HIV vectors packaged with an MLV integrase, indicates that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions in the genome. This study identifies TFBSs as differential genomic determinants of retroviral target site selection in the human genome, and suggests that transcription factors binding the LTR enhancer may synergize with the integrase in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions. Our data indicate that gamma-retroviruses and lentiviruses have evolved dramatically different strategies to interact with the host cell chromatin, and predict a higher risk in using gamma-retroviral vs. lentiviral vectors for human gene therapy applications.

2009 - Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design [Articolo su rivista]
Maruggi, G; Porcellini, S; Facchini, G; Perna, Sk; Cattoglio, C; Sartori, D; Ambrosi, A; Schambach, A; Baum, C; Bonini, C; Bovolenta, C; Mavilio, Fulvio; Recchia, Alessandra

The integration characteristics of retroviral (RV) vectorsincrease the probability of interfering with the regulationof cellular genes, and account for a tangible riskof insertional mutagenesis in treated patients. To assessthe potential genotoxic risk of conventional or self-inactivating(SIN) γ-RV and lentiviral (LV) vectors independentlyfrom the biological consequences of the insertionevent, we developed a quantitative assay based on realtimereverse transcriptase—PCR on low-density arraysto evaluate alterations of gene expression in individualprimary T-cell clones. We show that the Moloney leukemiavirus long terminal repeat (LTR) enhancer has thestrongest activity in both a γ-RV and a LV vector context,while an internal cellular promoter induces deregulationof gene expression less frequently, at a shorter range andto a lower extent in both vector types. Downregulationof gene expression was observed only in the context ofLV vectors.This study indicates that insertional gene activationis determined by the characteristics of the transcriptionalregulatory elements carried by the vector, andis largely independent from the vector type or design.

2008 - Absence of an intrathecal immune reaction to a helper-dependent adenoviral vector delivered into the cerebrospinal fluid of non-human primates. [Articolo su rivista]
Butti, E; Bergami, A; Recchia, Alessandra; Brambilla, E; Franciotta, D; Cattalini, A; Stornaiuolo, A; Lachapelle, F; Comi, G; Mavilio, Fulvio; Martino, G; Furlan, R.

Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration.

2008 - Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors [Articolo su rivista]
DI NUNZIO, Francesca; Maruggi, Giulietta; Stefano, Ferrari; Enzo Di, Iorio; Poletti, Valentina; Marta, Garcia; Marcela Del, Rio; DE LUCA, Michele; Fernando, Larcher; Pellegrini, Graziella; Mavilio, Fulvio

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer–restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.

2008 - Gene therapy of inherited skin adhesion disorders [Articolo su rivista]
De Luca, M.; Pellegrini, G.; Mavilio, F.

Gene therapy is a potential treatment for devastatinginherited diseases for which there is little hope offinding a conventional cure. These include lethal diseaseslike immunodeficiencies and metabolic disorders,and non-lethal conditions associated with poorquality of life and life-long symptomatic treatments,like muscular dystrophy, cystic fibrosis or thalassemia.Skin adhesion defects belong to both groups. For thenon-lethal forms, gene therapy, or transplantation ofcultured skin derived from genetically corrected epidermalstem cells, represents a very attractive therapeuticoption, and potentially a definitive treatment.Recent advances in gene transfer and stem cell culturetechnology are making this option closer than ever.This paper critically reviews the progress and prospectsof gene therapy for skin adhesion defects, andthe technical and non-technical factors currently limitingits development.

2008 - Genetic modification of somatic stem cells. The progress, problems and prospects of a new therapeutic technology [Articolo su rivista]
Mavilio, Fulvio; G., Ferrari

Not available

2008 - IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse models of multiple sclerosis [Articolo su rivista]
Butti, E; Bergami, A; Recchia, Alessandra; Brambilla, E; Del Carro, U; Amadio, S; Cattalini, A; Esposito, M; Stornaiuolo, A; Comi, G; Pluchino, S; Mavilio, Fulvio; Martino, G; Furlan, R.

Central nervous system (CNS) delivery of anti-inflammatory cytokines, such as interleukin 4 (IL4), holds promise as treatment for multiple sclerosis (MS). We have previously shown that short-term herpes simplex virus type 1-mediated IL4 gene therapy is able to inhibit experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in mice and non-human primates. Here, we show that a single administration of an IL4-expressing helper-dependent adenoviral vector (HD-Ad) into the cerebrospinal fluid (CSF) circulation of immunocompetent mice allows persistent transduction of neuroepithelial cells and long-term (up to 5 months) CNS transgene expression without toxicity. Mice affected by chronic and relapsing EAE display clinical and neurophysiological recovery from the disease once injected with the IL4-expressing HD-Ad vector. The therapeutic effect is due to the ability of IL4 to increase, in inflamed CNS areas, chemokines (CCL1, CCL17 and CCL22) capable of recruiting regulatory T cells (CD4+CD69-CD25+Foxp3+) with suppressant functions. CSF delivery of HD-Ad vectors expressing anti-inflammatory molecules might represent a valuable therapeutic option for CNS inflammatory disorders.

2008 - Medicina rigenerativa e nuove frontiere terapeutiche [Capitolo/Saggio]
Pellegrini, Graziella; Mavilio, Fulvio

Lo sviluppo delle colture cellulari ha messo in evidenza che le cellule adulte (non più allo stadio embrionale) potevano essere manipolate e dare origine non solo alla proliferazione, che ne aumentava il numero, ma anche al differenziamento che garantiva che quelle cellule potessero svolgere le funzioni più "specializzate" dei tessuti. Tutto ciò ha aperto prospettive completamente nuove alla medicina e si è fatta strada l'idea di costruire "pezzi di ricambio" o, comunque, imparare a trasmettere informazioni ad un tessuto danneggiato perché questo venisse riparato. La pionieristica chirurgia del cardiochirurgo C. Barnard ha dimostrato che il cuore di un individuo poteva essere trapiantato in un altro individuo; oggi questo concetto si sta sostituendo con l'idea che si può tentare di ricostruire un tessuto di un paziente in laboratorio, partendo dalle sue stesse cellule. L'uso di cellule "autologhe" (cioè dello stesso individuo e non di un donatore) per ricostruire o modificare le funzioni di un tessuto, evita tutti i problemi di rigetto e la tossicità dei farmaci che, normalmente, si somministrano per ridurre le reazioni di rigetto. La lista di tessuti che, potenzialmente, possono essere ricostruiti (o ingegnerizzati) è in crescita. Tutto ciò è dovuto in larga parte ai recenti progressi nello studio delle cellule staminali e alla individuazione delle caratteristiche biologiche uniche di queste cellule, sebbene non tutte le terapie basate sulle cellule staminali prevedano la ricostruzione del tessuto in vitro, come ad esempio alcune terapie che utilizzano le cellule staminali neuronali, ma piuttosto la stimolazione di cellule endogene. Altre terapie, inoltre, prevedono l'uso di cellule staminali geneticamente modificate per produrre sostanze con funzioni terapeutiche. Bisogna ricordare che, nonostante le aspettative, il passaggio alla applicazione clinica è stato raggiunto, ad oggi, solo in poche aree, in particolare in quelle nelle quali si era sviluppata una conoscenza più approfondita della biologia delle cellule staminali di quel distretto corporeo. I tessuti che oggi sono ricostruiti ed applicati con relativa facilità comprendono una ampia gamma di superfici epiteliali (pelle, cornea, congiuntiva e membrane mucose), tessuti scheletrici e sistema ematopoietico. Tra questi, quelli che sono stati geneticamente modificati ed utilizzati con successo per applicazione clinica, includono il sistema ematopoietico e gli epiteli. Questi sistemi sono intrinsecamente differenti nella loro capacità di autorinnovarsi, nella loro fisiologia e nella struttura fisica. La valutazione e lo studio delle diversità intrinseche ad ogni organo o sistema e la conoscenza della regolazione delle sue cellule staminali è essenziale per lo sviluppo di adeguate strategie di intervento clinico nei vari distretti corporei e nelle diverse patologie. Il capitolo descrive alcuni tra i diversi modelli possibili e alcune problematiche legate all'uso delle due branche principali della medicina rigenerativa: la terapia cellulare e la terapia genica.

2008 - Role of CD34 antigen in myeloid differentiation of human hematopoietic progenitor cells [Articolo su rivista]
Salati, Simona; Zini, Roberta; Bianchi, Elisa; Testa, Anna; Mavilio, Fulvio; Manfredini, Rossella; Ferrari, Sergio

CD34 is a transmembrane protein that is strongly expressed on hematopoietic stem/progenitor cells (HSCs); despite its importance as a marker of HSCs, its function is still poorly understood, although a role in cell adhesion has been demonstrated. To characterize the function of CD34 antigen on human HSCs, we examined, by both inhibition and overexpression, the role of CD34 in the regulation of HSC lineage differentiation. Our results demonstrate that CD34 silencing enhances HSC granulocyte and megakaryocyte differentiation and reduces erythroid maturation. In agreement with these results, the gene expression profile of these cells reveals the upregulation of genes involved in granulocyte and megakaryocyte differentiation and the downregulation of erythroid genes. Consistently, retroviral-mediated CD34 overexpression leads to a remarkable increase in erythroid progenitors and a dramatic decrease in granulocyte progenitors, as evaluated by clonogenic assay. Together, these data indicate that the CD34 molecule promotes the differentiation of CD34+ hematopoietic progenitors toward the erythroid lineage, which is achieved, at least in part, at the expense of granulocyte and megakaryocyte lineages.

2007 - C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells. [Articolo su rivista]
Barbaro, V; Testa, Anna; DI IORIO, E; Mavilio, Fulvio; Pellegrini, Graziella; DE LUCA, Michele

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein (C/EBP), Bmi1, and Np63 identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBP and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBP inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27Kip1 and p57Kip2. These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBP, but not Np63, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBP is recruited to the chromatin of positively (p27Kip1 and p57Kip2) and negatively (p16INK4A and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.

2007 - Hot spots of retroviral integrations in human CD34+ hematopoietic cells [Articolo su rivista]
Cattoglio, C; Facchini, Giulia; Sartori, D; Antonelli, A; Miccio, Annarita; Cassani, B; Schmidt, M; VON KALLE, C; Howe, S; Thrasher, A. J.; Aiuti, A; Ferrari, G; Recchia, Alessandra; Mavilio, Fulvio

Insertional oncogenesis is a possible consequence of the integration of gamma-retroviral (RV) or lentiviral (LV) vectors into the human genome. RV common insertion sites (CISs) have been identified in hematopoietic malignancies and in the nonmalignant progeny of transduced hematopoietic stem/progenitor cells (HSCs), possibly as a consequence of clonal selection in vivo. We have mapped a large number of RV and LV integrations in human CD34+ HSCs, transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for more than 21% of the RV integration events, while they are significantly less frequent in the case of LV vectors. RV but not LV hot spots are highly enriched in proto-oncogenes, cancer-associated CISs, and growth-controlling genes, indicating that at least part of the biases observed in the HSC progeny in vivo are characteristics of RV integration, already present in nontransplanted cells. Genes involved in hematopoietic and immune system development are targeted at high frequency and enriched in hot spots, suggesting that the CD34+ gene expression program is instrumental in directing RV integration. The lower propensity of LV vectors for integrating in potentially dangerous regions of the human genome may be a factor determining a better safety profile for gene therapy applications.

2007 - Long-term engraftment of single genetically modified epidermal stem cell-derived clones enables safety pre-assessment of human cutaneous gene therapy [Articolo su rivista]
Larcher, F; Dellambra, E; Rico, L; Bondanza, S; Murillas, R; Cattoglio, Claudia; Mavilio, Fulvio; Jorcano, J. L.; Zambruno, G; DEL RIO, M.

Predicting the risks of permanent gene therapy approaches involving the use of integrative gene-targeting vectors has become a critical issue after the unfortunate episode of a clinical trial in children with X-linked severe combined immunodeficiency (X-SCID). Safety pre-assessment of single isolated gene-targeted stem cells or their derivative clones able to regenerate their tissue of origin would be a major asset in addressing untoward gene therapy effects in advance. Human epidermal stem cells, which have extensive proliferative potential in vitro, theoretically offer such a possibility as a method of assessment. By means of optimized organotypic culture and grafting methods, we demonstrate the long-term in vivo regenerative capacity of single gene-targeted human epidermal stem cell clones (holoclones). Both histopathological analysis of holoclone-derived grafts in immunodeficient mice and retroviral insertion site mapping performed in the holoclone in vitro and after grafting provide proof of the feasibility of pre-assessing genotoxicity risks in isolated stem cells before transplantation into patients. Our results provide an experimental basis for previously untested assumptions about the in vivo behavior of epidermal stem cells prospectively isolated in vitro and pave the way for a safer approach to cutaneous gene therapy.

2007 - Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy [Articolo su rivista]
Aiuti, A; Cassani, B; Andolfi, G; Mirolo, M; Biasco, L; Recchia, Alessandra; Urbinati, Fabrizia; Valacca, C; Scaramuzza, S; Cazzola, M; Sartori, D; Ambrosi, A; DI SERIO, C; RONCAROLO M., G; Mavilio, Fulvio; AND BORDIGNON, C.

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase–deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34+ cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.

2007 - Transduction of human hematopoietic stem cells by lentiviral vectors pseudotyped with the RD114-TR chimeric envelope glycoprotein [Articolo su rivista]
DI NUNZIO, Francesca; Piovani, B; COSSET F., L; Mavilio, Fulvio; Stornaiuolo, A.

Lentiviral vectors are efficiently pseudotyped with RD114-TR, a chimeric envelope glycoprotein made of the extracellular and transmembrane domains of the feline leukemia virus RD114 and the cytoplasmic tail of the murine leukemia virus amphotropic envelope. RD114-TR-pseudotyped vectors may be concentrated by centrifugation, are resistant to complement inactivation, and are suitable for both ex vivo and in vivo gene therapy applications. We analyzed RD114-TR-pseudotyped, HIV-1-derived lentiviral vectors for their ability to transduce human cord blood, bone marrow, and peripheral blood mobilized CD34+ hematopoietic stem/progenitor cells. Transduction efficiency was analyzed in CD34+ cells in liquid culture, in CD34+ clonogenic progenitors in semisolid culture, and in CD34+ repopulating stem cells after xenotransplantation in NOD-SCID mice. Compared with a standard VSV-G-based packaging system, RD114-TR-pseudotyped particles transduced hematopoietic stem/progenitor cells at lower multiplicity of infection, with lower toxicity and less pseudo-transduction at comparable vector copy number per genome. Potential changes in the CD34+ cell transcription profile and phenotype on transduction with RD114-TR-pseudotyped vectors was comparatively investigated by microarray analysis. Our study shows that the biology of repopulating hematopoietic stem cells and their progeny is not affected by transduction with RD114-TR-pseudotyped lentiviral vectors. RD114-TR is compatible with the development of lentiviral stable packaging cell lines, and may become the envelope of choice for clinical studies aiming at safe and efficient genetic modification of human hematopoietic stem cells.

2006 - Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. [Articolo su rivista]
Mavilio, Fulvio; Pellegrini, Graziella; Ferrari, S.; DI NUNZIO, Francesca; Di Iorio, E.; Recchia, Alessandra; Maruggi, Giulietta; Ferrari, G.; Provasi, E.; Bonini, C.; Capurro, S.; Conti, A.; Magnoni, Cristina; Giannetti, Alberto; DE LUCA, Michele

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

2006 - Gene therapy in combination with tissue engineering to treat epidermolysis bullosa [Articolo su rivista]
S., Ferrari; Pellegrini, Graziella; T., Matsui; Mavilio, Fulvio; DE LUCA, Michele

Abstract:In the last 20 years epidermal stem cells have been extensively used for tissue regeneration of epidermis and other epithelial surfaces. The tremendous progress achieved has led to the development of protocols aimed at the correction of rare genetic disorders such as epidermolysis bullosa (EB), a severe, often lethal, blistering disorder of the skin. Approximately 400,000 – 500,000 people are affected worldwide and no definitive treatments have yet been developed. Gene therapy might represent an alternative therapeutic approach. This paper reviews the different strategies used to genetically modify keratinocytes from EB patients and addresses issues such as the use of in vivo or ex vivo approaches, how to target keratinocytes with stem cell properties in order to have long-term therapeutic gene expression, and which gene transfer agents should be used. The progress made has led the authors' group to submit a request for a Phase I/II ex vivo therapy clinical trial for patients with junctional EB.

2006 - Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells [Articolo su rivista]
Recchia, Alessandra; C., Bonini; Z., Magnani; Urbinati, Fabrizia; D., Sartori; S., Muraro; Tagliafico, Enrico; A., Bondanza; Mtl, Stanghellini; M., Bernardi; A., Pescarollo; F., Ciceri; C., Bordignon; Mavilio, Fulvio

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo, with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

2006 - Site-specific integration into the human genome: Ready for clinical application? [Articolo su rivista]
Recchia, Alessandra; Mavilio, Fulvio

Inserting genetic information at precise locations into the human genome has been the goal of the gene therapy community for almost two decades. Despite their spectacular progress in many fields of mammalian genetics, genome editing and homologous recombination are still too inefficient to be applied to human primary cells and tissues, the targets of any medical application. Site-specific integration, or the insertion of genes at known locations by enzymes that target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology.

2006 - Towards a gene therapy clinical trial for epidermolysis bullosa [Articolo su rivista]
Ferrari, S; Pellegrini, Graziella; Matsui, T; Mavilio, Fulvio; DE LUCA, Michele

Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the underneath derma lead to severe, often lethal, blistering disorders of the skin known as Epidermolysis Bullosa (EB). About 400000-500000 people worldwide are affected and no definitive treatments have yet been developed. Gene therapy might represent an alternative therapeutic approach for these devastating inherited disorders. In the last 10 years pre-clinical studies have shown that human epidermal stem cells can be stably transduced using integrating vectors allowing long-term genetic correction of the adhesion defects affecting EB keratinocytes both in vitro and in vivo after transplantation onto immunodeficient animals. In addition tremendous progress have been achieved in the clinical applications of cultured keratinocytes (cell therapy) for the regeneration of the epidermis over full thickness wounds or the restoration of damaged corneal surfaces. The combination of (i) optimised culturing conditions not altering the epidermal stemness, (ii) gene transfer vectors able to target epidermal stem cells very efficiently and (iii) surgical procedures allowing the grafting of large skin areas have therefore led our group to submit the first phase I/II gene therapy clinical trial for Junctional Epidermolysis Bullosa.

2005 - Competitive engraftment of hematopoietic stem cells genetically modified with a truncated erythropoietin receptor [Articolo su rivista]
URBINATI, Fabrizia; F., Lotti; G., Facchini; M., Montanari; G., Ferrari; MAVILIO, Fulvio; GRANDE, Alexis

Transplantation of genetically modified hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Engraftment of HSCs, however, requires toxic myeloablative treatments, which render this approach questionable for non-life-threatening disorders. A potential alternative is the use of transgenes, which allows positive selection of HSCs in vivo. We used retroviral vectors to express a truncated derivative of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. Murine HSCs expressing tEpoR at different levels (1500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into fully or partially myeloablated co-isogenic mouse recipients. Long-term analysis of transplanted mice showed that expression of tEpoR at paraphysiological levels (similar to 1500 receptors/cell) has no effect on steady-state hematopoiesis and induces no further expansion of transduced cells after the engraftment period. Human cord blood-derived CD34(+) stem/progenitor cells transduced with a lentiviral vector expressing tEpoR expand their clonogenic capacity in vitro, and significantly increase their marrow repopulation capacity upon xenotransplantation into sublethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival, and differentiation properties. These data indicate that expression of tEpoR is an effective strategy to promote selective engraftment of genetically modified HSCs upon transplantation in both myeloablative and nonmyeloablative conditions, without the use of toxic drugs for selection.

2005 - Gene therapy approaches for epidermolysis bullosa [Articolo su rivista]
S., Ferrari; Pellegrini, Graziella; Mavilio, Fulvio; DE LUCA, Michele

Human epidermis consists of a stratified epithelium mainly composed of keratinocytes and relies on a stem cell compartment to undergo constant regeneration, Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the epidermal basement membrane lead to severe. and very often lethal, blistering disorders known as epidermolysis bullosa. Gene therapy represents a promising potential treatment for these devastating inherited disorders. Human epidermal stem cells can be cultivated ex vivo and stably transduced with integrating gene transfer vectors. allowing genetic and, more important, phenotypic correction of the adhesion properties of keratinocytes, Here we will review some of the issues that need to be addressed to make gene therapy a realistic treatment for these disorders, such as (1) which cells should be targeted, (2) which approach (in vivo or ex vivo) should be chosen, and (3) which gene transfer vector (retrovirus, lentivirus, or integrating nonviral strategies,) should be used for stable gene correction. In the last 10 years, many reports have shown that gene transfer approaches to target epidermal stem cells are feasible and able to restore the adhesion properties of primary keratinocytes from patients with epidermolysis bullosa. In addition, tremendous progress has been achieved in culturing epidermal stem cells and generating sheets of stratified epithelium tor permanent coverage of full-thickness bums. Gene modification of stem cells in combination with advanced tissue-engineering techniques could therefore represent a realistic option for patient-4 with epidermolysis bullosa.

2005 - Genetic modification of human hematopoietic stem cells [Articolo su rivista]
Mavilio, F.

Transplantation of genetically modified hematopoietic stem cells is a potential therapy for a variety of genetic and acquired blood disorders, such as severe combined immunodeficiencies, thalassemia and AIDS. Genetic modification of stem cells can be carried out ex vivo, by transducing bone marrow or peripheral blood stem cell-rich fractions with viral vectors carrying therapeutic genes. These vectors must be able to transduce long-term repopulating stem cells, and allow appropriate transgene expression. Recent improvements in cell culture and vector technology are providing new tools for obtaining clinically relevant numbers of genetically modified hematopoietic stem cells from a standard bone marrow harvest. The proof of the therapeutic potential of this technology is the successful therapy of two different forms of severe combined immunodeficiencies, rare genetic disorders associated with recurrent infections that may be fatal in the first years of life. These pivotal trials, however, have also uncovered the oncogenic potential of random retroviral insertions into the human genome. For non life-threatening disorders, the risk of insertional oncogenesis may be unacceptable, and new research is needed to develop new, safer and more efficient gene transfer vectors for clinical application.

2005 - Stem cell plasticity: time for a reappraisal? [Articolo su rivista]
Rm, Lemoli; F., Bertolini; R., Cancedda; DE LUCA, Michele; A., DEL SANTO; G., Ferrari; Ferrari, Sergio; G., Martino; Mavilio, Fulvio; S., Tura

n recent years an increasing number of publications have claimed that adult mammalian stem cells (SC) may be capable of differentiating across tissue lineage boundaries and that this plasticity may represent a novel therapeutic strategy for tissue regeneration. However, after a first phase of excitement, the issue of somatic SC plasticity remains controversial and the therapeutic perspectives are still elusive. In this review, we examine the general mechanisms which govern the function of SC, the identification and functional characterization of adult SC of different tissues and their putative capacity to transdifferentiate into mature cells of different origin. The potential clinical applications of adult SC for regenerative medicine are also discussed in each chapter. The method employed for preparing this review was the informal consensus development. Members of the Working Group on SC met four times and discussed the single points, previously assigned by the Chairman (S.T.), in order to achieve an agreement on different opinions and approve the final manuscript. All the authors of the present review have been working in the field of SC and have contributed original papers to peer-reviewed journals. In addition to the authors' own work, the present review examines articles published in journals covered by the Science Citation Index and Medline.

2005 - T Lymphocytes transduced with a lentiviral vector expressing F12-Vif are protected from HIV-1 infection in an APOBEC3G-independent manner [Articolo su rivista]
Vallanti, G.; Lupo, R.; Federico, M.; Mavilio, Fulvio; Bovolenta, C.

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.

2004 - Selective engraftment of genetically modified hematopoietic stem cells by a truncated erythropoietin receptor [Relazione in Atti di Convegno]
Urbinati, F; Grande, Alexis; Lotti, F; Facchini, Gl; Ferrari, G; Mavilio, Fulvio

Genetic modification of hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Transplantation of HSCs, however, requires toxic myeloablation regimens which render this approach questionable for non life-threatening disorders. A potential alternative is the use of transgenes allowing positive selection of HSCs in vivo. We used MLV-derived retroviral vectors and HIV-derived lentiviral vectors to express a truncated form of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. The tEpoR molecule carries a deletion of the 91 carboxy-terminal amino acids, which enhances its proliferative response due to the elimination of a negative regulatory domain. Murine HSCs expressing retrovirally-transferred tEpoR at different levels (1,500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into co-isogenic mouse recipients. Human cord blood-derived CD34+ stem/progenitor cells transduced with a lentiviral vector expressing tEpoR significantly increase their marrow repopulation capacity upon xenotransplantation into sub-lethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival and differentiation properties. Long-term analysis of serially transplanted mice showed that expression of tEpoR at physiological levels (i.e., comparable with, or slightly higher than, those of the wild-type EpoR in erythroblastic cells) has no effect on steady-state hematopoiesis, and induces no further expansion of transduced cells after the engraftment period. However, significant overexpression of tEpoR (&gt;8-fold the physiological levels) causes mild anemia and erythrocyte morphological abnormalities. These data indicate that expression of tEpoR is a potential alternative for in vivo selection of murine and human repopulating HSCs.

2004 - Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors [Articolo su rivista]
Recchia, Alessandra; L., Perani; D., Sartori; L., Olgiati; Mavilio, Fulvio

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivo by the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications.

2004 - The future of gene therapy [Articolo su rivista]
M., Cavazzana Calvo; A., Thrasher; Mavilio, Fulvio

Gene therapy has the potential to treat devastating inherited diseases for which there is little hope of finding a conventional cure. In the late 1990s, our groups in Paris, London and Milan began treating children suffering from rare immune disorders (severe combined immunodeficiencies, or SCIDs). The successful treatment of the first patients was greeted with excitement when it was first reported in 2000 and 2002. Sadly, this euphoria turned to alarm at the end of 2002, when two of the ten children treated in France developed leukaemia-like conditions.

2004 - Towards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type II [Abstract in Rivista]
Ferrari, S; Ferrari, G; Rossi, C; Miccio, Annarita; Volpi, Nicola; Mavilio, Fulvio; Pellegrini, Graziella; De, Luca

Towards the correction of genetic defect in corneal keratinocytes from patient with macular corneal Dystrophy type II

2003 - French gene therapy group reports on the adverse event in a clinical trial of gene therapy for X-linked severe combined immune deficiency (X-SCID). Position statement from the European Society of Gene Therapy (ESGT) [Articolo su rivista]
Gansbacher, B.; Danos, O.; Dickson, G.; Thielemans, K.; Cosset, F. -L.; Deglon, N.; Dilber, M. S.; Galun, E.; Klatzmann, D.; Mavilio, F.; Taylor, N.

2003 - Safety of retroviral gene marking with a truncated NGF receptor [Articolo su rivista]
Bonini, C; Grez, M; Traversari, C; Ciceri, F; Martkel, S; Ferrari, G; Dinauer, M; Sadat, M; Aiuti, A; Deola, S; Raddrizzani, M; Hagenbeek, A; Apperley, J; Ebeling, S; Martens, A; Kolb, Hj; Weber, M; Lotti, F; Grande, Alexis; Weissinger, E; Bueren, Ja; Lamana, M; Falkenburg, Jhf; Heemskeerk, Mhm; Austin, T; Kornblau, S; Marini, F; Benati, C; Magnani, Z; Cazzaniga, S; Toma, S; GALLO STAMPINO, C; Introna, M; Slavin, S; Greenberg, Pd; Bregni, M; Mavilio, Fulvio; Bordignon, C.

Random integration into the host cell genome and inappropriate transgene expression are major safety concerns for the clinical use of retroviral vectors.

2003 - The choice of a suitable lentivirus vector: transcriptional targeting [Capitolo/Saggio]
Lotti, F.; Mavilio, F.

2002 - Muscle-derived hematopoietic stem cells are hematopoietic in origin [Articolo su rivista]
SL McKinney, Freeman; Ka, Jackson; Fd, Camargo; G., Ferrari; Mavilio, Fulvio; Ma, Goodell

It has recently been shown that mononuclear cells from murine skeletal muscle contain the potential to repopulate all major peripheral blood lineages in lethally irradiated mice, but the origin of this activity is unknown. We have fractionated muscle cells on the basis of hematopoietic markers to show that the active population exclusively expresses the hematopoietic stem cell antigens Sca-1 and CD45. Muscle cells obtained from 6- to 8-week-old C57BL/6-CD45.1 mice and enriched for cells expressing Sca-1 and CD45 were able to generate hematopoietic but not myogenic colonies in vitro and repopulated multiple hematopoietic lineages of lethally irradiated C57BL/6-CD45.2 mice. These data show that muscle-derived hematopoietic stem cells are likely derived from the hematopoietic system and are a result not of transdifferentiation of myogenic stem cells but instead of the presence of substantial numbers of hematopoietic stem cells in the muscle. Although CD45-negative cells were highly myogenic in vitro and in vivo, CD45-positive muscle-derived cells displayed only very limited myogenic activity and only in vivo.

2002 - Myogenic stem cells from the bone marrow: a therapeutic alternative for muscular dystrophy? [Articolo su rivista]
Ferrari, G; Mavilio, Fulvio

Differentiated muscle fibres can be formed by transplanted haematopoietic stem cells in models of acute or chronic muscle regeneration, including the dystrophin-deficient mdx mouse. Muscle-forming activity can be found in adult, foetal and embryonic haematopoietic tissues. The blood-to-muscle transition may be due to transdifferentiation of haematopoietic progenitors in response to local signals provided by the regenerating muscle. These signals are only poorly provided by the muscle of the mdx mouse, since transplantation into these mice of normal C57Bl/6 bone mar-row gives rise only to a minimal number of muscle fibres expressing the normal dystrophin protein (<1%) throughout the animal life span. Expansion and active recruitment to myogenic differentiation of transplanted haematopoietic cells are therefore critical factors for a future use of bone marrow transplantation in cell/gene therapy of muscular dystrophy. (C) 2002 Elsevier Science B.V. All rights reserved.

2002 - Toward gene therapy of junctional epidermolysis bullosa (JEB) [Abstract in Atti di Convegno]
DE LUCA, Michele; Dellambra, E; Pellegrini, Graziella; Guerra, L; Bondanza, S; Mavilio, Fulvio

Non disponibile

2002 - Transcriptional targeting of lentiviral vectors by LTR enhancer replacement [Articolo su rivista]
Lotti, F.; Menguzzato, E.; Rossi, C.; Naldini, L.; Ailles, L.; Mavilio, Fulvio; Ferrari, G.

Gene therapy of many genetic diseases requires permanent gene transfer into self-renewing stem cells and restriction of transgene expression to specific progenies. Human immunodeficiency virus (HIV)-derived lentiviral vectors are very effective in transducing rare, nondividing stem cell populations (e.g., hematopoietic stem cells) without altering their long-term repopulation and differentiation capacities. We developed a strategy for transcriptional targeting of lentiviral vectors based on replacing the viral long terminal repeat (LTR) enhancer with cell lineage-specific, genomic control elements. An upstream enhancer (HS2) of the erythroid-specific GATA-1 gene was used to replace most of the U3 region of the LTR, immediately upstream of the HIV type 1 (HIV-1) promoter. The modified LTR was used to drive the expression of a reporter gene (the green fluorescent protein [GFP] gene), while a second gene (a truncated form of the p75 nerve growth factor receptor [DeltaLNGFR]) was placed under the control of an internal constitutive promoter to monitor cell transduction, or to immunoselect transduced cells, independently from the expression of the targeted promoter. The transcriptionally targeted vectors were used to transduce cell lines, human CD34+ hematopoietic stem-progenitor cells, and murine bone marrow (BM)-repopulating stem cells. Gene expression was analyzed in the stem cell progeny in vitro and in vivo after xenotransplantation into nonobese diabetic-SCID mice or BM transplantation in coisogenic mice. The modified LTR directed high levels of transgene expression specifically in mature erythroblasts, in a TAT-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the modified LTR was higher, better restricted, and showed less position- effect variegation than that obtained by the same combination of enhancer-promoter elements placed in a conventional, internal position. Cloning of the woodchuck hepatitis virus posttranscriptional regulatory element at a defined position in the targeted vector allowed selective accumulation of the genomic transcripts with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of a spliced, major viral transcript to express a therapeutic gene and that of an internal, independently regulated promoter to express an additional gene for either cell marking or in vivo selection purposes.

2002 - Transcriptional targeting of lentiviral vectors by long terminal repeat enhancer replacement [Articolo su rivista]
Lotti, F.; Menguzzato, E.; Rossi, C.; Naldini, L.; Ailles, L.; Mavilio, F.; Ferrari, G.

Gene therapy of many genetic diseases requires permanent gene transfer into self-renewing stem cells and restriction of transgene expression to specific progenies. Human immunodeficiency virus (HIV)-derived lentiviral vectors are very effective in transducing rare, nondividing stem cell populations (e.g., hematopoietic stem cells) without altering their long-term repopulation and differentiation capacities. We developed a strategy for transcriptional targeting of lentiviral vectors based on replacing the viral long terminal repeat (LTR) enhancer with cell lineage-specific, genomic control elements. An upstream enhancer (HS2) of the erythroid-specific GATA-1 gene was used to replace most of the U3 region of the LTR, immediately upstream of the HIV type 1 (HIV-1) promoter. The modified LTR was used to drive the expression of a reporter gene (the green fluorescent protein [GFP] gene), while a second gene (a truncated form of the p75 nerve growth factor receptor [ΔLNGFR]) was placed under the control of an internal constitutive promoter to monitor cell transduction, or to immunoselect transduced cells, independently from the expression of the targeted promoter. The transcriptionally targeted vectors were used to transduce cell lines, human CD34+ hematopoietic stemprogenitor cells, and murine bone marrow (BM)-repopulating stem cells. Gene expression was analyzed in the stem cell progeny in vitro and in vivo after xenotransplantation into nonobese diabetic-SCID mice or BM transplantation in coisogenic mice. The modified LTR directed high levels of transgene expression specifically in mature erythroblasts, in a TAT-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the modified LTR was higher, better restricted, and showed less position-effect variegation than that obtained by the same combination of enhancer-promoter elements placed in a conventional, internal position. Cloning of the woodchuck hepatitis virus posttranscriptional regulatory element at a defined position in the targeted vector allowed selective accumulation of the genomic transcripts with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of a spliced, major viral transcript to express a therapeutic gene and that of an internal, independently regulated promoter to express an additional gene for either cell marking or in vivo selection purposes.

2001 - Bone-marrow transplantation - Failure to correct murine muscular dystrophy [Articolo su rivista]
G., Ferrari; A., Stornaiuolo; Mavilio, Fulvio

Bone-marrow cells have the potential to differentiate into other cell types such as muscle fibres, and can be transplanted into acutely or chronically damaged muscle as a way of delivering normal dystrophin (the protein that is defective or missing in Duchenne’s muscular dystrophy) to the skeletal and heart muscle of mdx mice, an animal model for this disease. But the corrective potential of this approach has been hard to estimate against the high background of muscle fibres that spontaneously revert to synthesizing dystrophin, a feature of the original mdx mutation. Here we test the long-term efficacy of bonemarrow transplantation in a different mdx mutant which is free of this problem and find that it has no impact on murine muscular dystrophy.

2001 - High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors [Articolo su rivista]
F., Moreau Gaudry; P., Xia; G., Jiang; Np, Perelman; G., Bauer; J., Ellis; Kh, Surinya; Mavilio, Fulvio; Ck, Shen; P., Malik

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self-inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [18] enhancers) and WPRE expressed at levels higher than the HS2/beta -promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34+ cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The 18/HS-40-containing vector encoding a hybrid human beta/gamma -globin gene led to 43% to 113% human gamma -globin expression/copy of the mouse alpha -globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to Identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.

2001 - The recruitment of SOX/OCT complexes and the differential activity of HOXA1 and HOXB1 modulate the Hoxb1 auto-regulatory enhancer function [Articolo su rivista]
G., Di Rocco; A., Gavalas; H., Popperl; R., Krumlauf; Mavilio, Fulvio; Zappavigna, Vincenzo

Regionally restricted expression patterns of Hox genes in developing embryos rely on auto-, cross-, and para-regulatory transcriptional elements. One example is the Hoxb1 auto-regulatory element (b1-ARE), which drives expression of Hoxb1 in the fourth rhombomere of the hindbrain, We previously showed that HOXB1 and PBX1 activate transcription from the b1-ARE: by binding to sequences required for the expression of a reporter gene in rhombomere 4 in vivo, We now report: that in embryonal carcinoma cells, which retain characteristics of primitive neuroectodermal cells, the b1-ARE displays higher basal and HOX/PBX-induced activities than in other cell backgrounds. We have identified a bipartite-binding site for SOX/OCT heterodimers within the b1-ARE that accounts for its cell context-specific activity and is required for maximal transcriptional activity of HOX/PBX complexes in embryonal carcinoma cells. Furthermore, we found that in an embryonal carcinoma cell background, HOXB1 has a significantly higher transcriptional activity than its paralog HOXA1. We map the determinants for this differential activity within the HOXB1 N-terminal transcriptional activation domain. By using analysis in transgenic and HOXA1 mutant mice, we extended these findings on the differential activities of HOXA1 and HOXB1 in vivo, and we demonstrated that they are important for regulating aspects of HOXB1 expression in the hindbrain. We found that mutation of the SOX/OCT site and targeted inactivation of Hoxa1 both impair the response of the b1-ARE to retinoic acid in transgenic mice. Our results show that Hoxa1 is the primary mediator of the response of b1-ARE to retinoic acid in vivo and that this function is dependent on the binding of SOX/OCT heterodimers to the b1-ARE. These results uncover novel functional differences between Hox paralogs and their modulators.

2000 - Myogenic stem cells for the therapy of primary myopathies: wishful thinking or therapeutic perspective? [Articolo su rivista]
Cossu, G.; Mavilio, Fulvio

Primary myopathies are characterized by a progressive wasting of skeletal muscle that leads to deterioration of movements and, in the most severe cases, such as in Duchenne’s muscular dystrophy (DMD), to complete paralysis and death. Most myopathies in which the molecular defect has been identified are due to mutations affecting proteins that form a supramolecular link between the cytoskeleton and the extracellular matrix, such as dystrophin, the mutated protein in DMD. In the absence of one of these proteins, mechanical stress associated with contraction progressively leads to degeneration of the muscle fiber, although the underlying mechanisms are still poorly understood. In the first phase of the disease, new muscle fibers are formed by fusion of resident myoblasts, called satellite cells, which also bear the molecular defect of the fibers that they replace, and hence undergo the same fate. Once the proliferation potential of satellite cells is exhausted, there is no further regeneration and the skeletal muscle is replaced by connective tissue

2000 - Toward epidermal stem cell-mediated ex vivo gene therapy of junctional epidermolysis bullosa [Articolo su rivista]
E., Dellambra; Pellegrini, Graziella; L., Guerra; G., Ferrari; G., Zambruno; Mavilio, Fulvio; DE LUCA, Michele

Junctional epidermolysis bullosa (JEB) is a group of severe, inherited skin diseases caused by mutations in the genes encoding laminin 5 or other components of the hemidesmosome. Since human epidermis is a self-renewing tissue, gene therapy of JEB requires the stable integration of the transgene into the genome of the epidermal stem cell. Human epidermal stem cells can indeed be cultivated and stably transduced with replication-defective retroviral vectors, allowing full phenotypic correction of the adhesion properties of JEB keratinocytes. Epidermal stem cells generate cohesive sheets of stratified epithelium suitable for the permanent coverage of massive skin defects, and genetically modified epidermal sheets maintain long-term expression of the transgene after transplantation on immunodeficient animals. Moreover, we have developed a clinical procedure that allows transplantation of cultured epidermal sheets on large body areas under local anesthesia and without cicatricial outcomes. Thus, (1) the possibility of cultivating lining epithelia, (2) the availability of noninvasive surgical procedures that allow the grafting of large skin areas, and (3) the demonstration of sustained transgene expression in vitro and in vivo by epidermal stem cells, prompt us to propose the implementation of a phase I/II clinical trial aimed at the ex vivo gene therapy of selected JEB patients. The aim of the trial is to validate the ex vivo procedure in a clinical setting, to prove its overall safety, and to analyze critical issues such as long-term survival of the genetically modified implant, immune response against the transgene product, and persistence of transgene expression at therapeutic levels.PMID: 11084687 [PubMed - indexed for MEDLINE]

1999 - Reversible immortalization of human myogenic cells by site-specific excision of a retrovitally transferred oncogene [Articolo su rivista]
Berghella, L.; De Angelis, L.; Coletta, M.; Berarducci, B.; Sonnino, C.; Salvatori, G.; Anthonissen, C.; Cooper, R.; Butler-Browne, G. S.; Mouly, V.; Ferrari, G.; Mavilio, F.; Cossu, G.

Myogenic cells have a limited life span in culture, which prevents expansion at clinically relevant levels, and seriously limits any potential use in cell replacement or ex vivo gene therapy. We developed a strategy for reversibly immortalizing human primary myogenic cells, based on retrovirus- mediated integration of a wild-type SV40 large-T antigen (Tag), excisable by means of the Cre-Lox recombination system. Myogenic cells were transduced with a vector (LTTN-LoxP) expressing the SV40 Tag under the control of an LTR modified by the insertion of a LoxP site in the U3 region. Clonal isolates of Tag-positive cells showed modified growth characteristics and a significantly extended life span, while maintaining a full myogenic potential. Transient expression of Cre recombinase, delivered by transfection or adenoviral vector transduction, allowed excision of the entire provirus with up to &gt;90% efficiency. Cultures of Cre-treated (Tag-) or untreated (Tag+) myogenic cells were genetically labeled with a lacZ retroviral vector, and injected into the regenerating muscle of SCID/bg immunodeficient mice. Tag- cells underwent terminal differentiation in vivo, giving rise to clusters of β- Gal+ hybrid fibers with an efficiency comparable to that of control untransduced cells. Tag+ cells could not be detected after injection. Neither Tag+ nor Tag- cells formed tumor in this xenotransplantation model. Reversible immortalization by Tag therefore allows the expansion of primary myogenic cells in culture without compromising their ability to differentiate in vivo, and could represent a safe method by which to increase the availability of these cells for clinical application.

1999 - The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH [Articolo su rivista]
Berthelsen, J.; Kilstrup-Nielsen, C.; Blasi, F.; Mavilio, F.; Zappavigna, V.

Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. We studied the subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which they are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. We propose that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export.

1999 - Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells [Articolo su rivista]
Grande, Alexis; B., Piovani; A., Aiuti; S., Ottolenghi; Mavilio, Fulvio; G., Ferrari

Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.PMID: 10233879 [PubMed - indexed for MEDLINE]

1998 - Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-Binding context [Articolo su rivista]
Ma, Vigano'; G., DI ROCCO; Zappavigna, Vincenzo; Mavilio, Fulvio

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

1998 - High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies [Articolo su rivista]
Lattanzi, L.; Salvatori, G.; Coletta, M.; Sonnino, C.; De Angelis, M. G. C.; Gioglio, L.; Murry, C. E.; Kelly, R.; Ferrari, G.; Molinaro, M.; Crescenzi, M.; Mavilio, F.; Cossu, G.

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat- promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to β- galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.

1998 - Muscle regeneration by bone marrow-derived myogenic progenitors [Articolo su rivista]
Ferrari, G.; Cusella-De Angelis, G.; Coletta, M.; Paolucci, E.; Stornaiuolo, A.; Cossu, G.; Mavilio, F.

Growth and repair of skeletal muscle are normally mediated by the satellite cells that surround muscle fibers. In regenerating muscle, however, the number of myogenic precursors exceeds that of resident cells, implying migration or recruitment of undifferentiated progenitors from other sources. Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrate into areas of induced muscle degeneration, undergo myogenic differentiation, and participate in the regeneration of the damaged fibers. Genetically modified, marrow-derived myogenic progenitors could potentially be used to target therapeutic genes to muscle tissue, providing an alternative strategy for treatment of muscular dystrophies.

1998 - Prep1, a novel functional partner of Pbx proteins [Articolo su rivista]
Berthelsen, J.; Zappavigna, V.; Mavilio, F.; Blasi, F.

The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. We reported previously that purified UEF3 is a complex of several subunits. In this paper we report the cloning of the cDNA of one of the subunits which encodes for a novel human homeodomain protein, which we have termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains, is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. We further identify the other UEF3 subunit as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity towards the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo.

1998 - The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity [Articolo su rivista]
Berthelsen, J.; Zappavigna, V.; Ferretti, E.; Mavilio, F.; Blasi, F.

The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity, Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

1997 - A novel homeo-domain protein, prep 1, forms a regulatory complex with pbx proteins in vivo [Articolo su rivista]
Berthelsen, J.; Zappavigna, V.; Mavilio, F.; Blasi, F.

Expression of the human urokinase (uPA) gene is regulated by an enhancer containing a combined PEA3/AP-1 site and an AP-1 site, spaced 72bp apart. These two sites have been shown to synergistically cooperate in both basal level and PMA induced activity of the enhancer, dependent on an element, the co-operation mediator (COM) element, positioned between the two sites, and which bind several different nuclear factors. uPA Enhancer Factor 3 (UEF3) has been identified in various cell lines binding specifically to a site within the COM element and in addition, binds to similar regulating motifs found in various other promoters, showing similar organization and activity as the uPA enhancer. We have purified UEF3 from HeLa cell nuclei by standard chromatography. Purification data show that UEF3 is a complex formed by three polypeptides, p64, p50 and p40. Overall, the data further suggest that UEF3 is a heterodimer consiting of p64 complexed with either p50 orp40. We have cloned the cDNA p64, which encodes for a novel protein, termed Prepl, containing a homeodomain similar to those found in mammalian PBX and Drosophila Extradenticle (exd) homeo domain proteins and in yeast Mat2a protein. We find that Prepl exist as a stable complex with Pbx proteins in vivo and that this complex can be made in vitro reconstituting UEF3 activity. Pbx are proteins known to functionally interact with Hox proteins during development. Both its structure and its way of interacting with Pbx, show that Prepl is different from Hox proteins, and our data point to a novel regulatory role for Pbx/Prepl complexes proteins independent on Hox.

1997 - Functional dissection of a transcriptionally active, target specific Hox/Pbx complex. [Articolo su rivista]
G., DI ROCCO; Mavilio, Fulvio; Zappavigna, Vincenzo

breve descrizione

1997 - HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia [Articolo su rivista]
Bonini, C.; Ferrari, G.; Verzeletti, S.; Servida, P.; Zappone, E.; Ruggieri, L.; Ponzoni, M.; Rossini, S.; Mavilio, F.; Traversari, C.; Bordignon, C.

In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GvHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced. With the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GvHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.

1996 - A replication-deficient human immunodeficiency virus-1 genome as an interference-inducing provirus [Articolo su rivista]
Federico, M.; Nappi, F.; Mavilio, F.; Verani, P.

1996 - Anti-HIV Viral Interference Induced by Retroviral Vectors Expressing a Nonproducer HIV-1 Variant [Articolo su rivista]
Federico, M.; Bona, R.; D'Aloja, P.; Baiocchi, M.; Pugliese, K.; Nappi, F.; Chelucci, C.; Mavilio, F.; Verani, P.

A Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus (HIV-1) variant shows complete resistance to HIV-1 or HIV-2 superinfection. The F12-HIV provirus produces an altered HIV-1 protein pattern and cannot generate even immature viral particles. We demonstrated that HeLa CD4+ cells transfected with the F12-HIV genome resist HIV superinfection through a CD4-independent mechanism. As F12-HIV appears to be a useful system to induce anti-HIV intracellular immunization, we constructed various retroviral vectors containing the F12-HIV genome, modified by elimination of the F12 3 LTR and part of its nef gene, inserted ‘antisense’ with respect to the Moloney murine leukemia virus 5' LTR. Here we show that recombinant retroviral particles carrying the N2/F12-HIV nef- (as) construct can stably transduce both CEMss human cells and primary human peripheral blood lymphocytes, inducing the expression of the F12-HIV genome. These results could open the way to an anti-AIDS gene therapy strategy based on F12-HIV-induced intracellular immunization. © 1996 S. Karger AG, Basel.

1996 - Clonal analysis of stably transduced human epidermal stem cells in culture. [Articolo su rivista]
Mb, Mathor; G., Ferrari; E., Dellambra; M., Cilli; Mavilio, Fulvio; R., Cancedda; DE LUCA, Michele

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.

1996 - HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation [Articolo su rivista]
Zappavigna, V.; Falciola, L.; Citterich, M. H.; Mavilio, F.; Bianchi, M. E.

High mobility group protein 1 (HMG1) is a nonhistone, chromatin-associated nuclear protein with a proposed role in the regulation of eukaryotic gene expression. We show that HMG1 interacts with proteins encoded by the HOX gene family by establishing protein-protein contacts between the HMG box domains and the HOX homeodomain. The functional role of these interactions was studied using the transcriptional activity of the human HOXD9 protein as a model, HMG1 enhances, in a dose-dependent fashion, the sequence-specific DNA binding activity in vitro, and the transcriptional activation in a co-transfection assay in vivo, of the HOXD9 protein. Functional interaction between HMG1 and HOXD9 is dependent on the DNA binding activity of the homeodomain, and requires the HOXD9 transcriptional activation domain, HMG1 enhances activation by HOXD9, but not by HOXD8, of the HOXD9-controlled element. Specific target recognition and functional interaction with HMG1 can be transferred to HOXD8 by homeodomain swapping. We propose that HMG1-like proteins might be general co-factors in HOX-mediated transcriptional activation, which facilitate access of HOX proteins to specific DNA targets, and/or introduce architectural constraints in the assembly of HOX-containing transcriptional complexes.

1996 - In vitro and transgenic analysis of a human HOXD4 retinoid-responsive enhancer [Articolo su rivista]
Morrison, A.; Moroni, M. C.; Ariza-McNaughton, L.; Krumlauf, R.; Mavilio, F.

Expression of vertebrate Hox genes is regulated by retinoids in cell culture and in early embryonic development. We have identified a 185-bp retinoid-responsive transcriptional enhancer 5' of the human HOXD4 gene, which regulates inducibility of the gene in embryonal carcinoma cells through a pattern of DNA-protein interaction on at least two distinct elements. One of these elements contains a direct repeat mediating ligand-dependent interaction with retinoic acid receptors, and is necessary though not sufficient for the enhancer function. The HOXD4 enhancer directs expression of a lacZ reporter gene in the neural tube of transgenic mouse embryos in a time-regulated and regionally restricted fashion, reproducing part of the anterior neuroectodermal expression pattern of the endogenous Hoxd-4 gene. Administration of retinoic acid to developing embryos causes alterations in the spatial restriction of the transgene expression domain, indicating that the HOXD4 enhancer is also a retinoid-responsive element in vivo. The timing of the retinoic acid response differs from that seen with more 3' Hox genes, in that it occurs much later. This shows that the temporal window of competence in the ability to respond to retinoic acid differs between Hox genes and can be linked to specific enhancers. Mutations in the direct repeat or in a second element in the enhancer affect both retinoid response in culture and developmental regulation in embryos, suggesting that co-operative interactions between different factors mediate the enhancer activity. These data provide further support for a role of endogenous retinoids in regulation and spatial restriction of Hox gene expression in the central nervous system.

1995 - A Retroviral Vector Containing a Muscle-Specific Enhancer Drives Gene Expression Only in Differentiated Muscle Fibers [Articolo su rivista]
Ferrari, G.; Salvatori, G.; Rossi, C.; Cossu, G.; Mavilio, F.

Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial β-galactosidase (β-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. β-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue. © 1995, Mary Ann Liebert, Inc. All rights reserved.

1995 - A nonproducer, interfering human immunodeficiency virus (HIV) type 1 provirus can be transduced through a murine leukemia virus-based retroviral vector: Recovery of an anti-HIV mouse/human pseudotype retrovirus [Articolo su rivista]
Federico, M.; Nappi, F.; Ferrari, G.; Chelucci, C.; Mavilio, F.; Verani, P.

The expression of a human immunodeficiency virus (HIV) type 1 provirus (F2-HIV) cloned from a nonproducer, chronically infected CD4 down-regulated Hut-78 cell clone (F12) does not lead to the formation of viral particles and, upon transfection in HeLa CD4+ cells, confers resistance to HIV superinfection without affecting the CD4 receptor exposure. In an attempt to transfer the anti-HIV properties of F12-HIV into human primary cells, we constructed a Moloney murine leukemia virus-based retroviral vector containing an F12-HIV genome lacking the 3' long terminal repeat and part of the nef gene, which was expressed under the control of its 5' long terminal repeat. The F12-HIV genome was inserted in the orientation opposite to that of the murine leukemia virus transcriptional unit and was designated the N2/F12-HIV nef- antisense vector. Lymphoblastoid CEMss cells, as well as human peripheral blood lymphocytes, were successfully transduced by the recombinant retrovirus emerging from the producer PA317 clones. CEMss clones expressing the F12-HIV nef-antisense vector became resistant to HIV superinfection even at the highest utilized multiplicity of infection (105 50% tissue culture infective doses per 106 cells). In transduced CEMss cells the viral interference induced by the F12-HIV expression is not due to CD4 HIV receptor down-regulation. Nonproducer, interfering HIV proviruses transduced into retroviral vectors may, therefore, provide an alternative strategy for the protection of CD4+ human primary cells from HIV infection, which strategy may be used in designating a safe and efficient gene therapy protocol for patients with AIDS.

1995 - Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients [Articolo su rivista]
Bordignon, C.; Notarangelo, L. D.; Nobili, N.; Ferrari, G.; Casorati, G.; Panina, P.; Mazzolari, E.; Maggioni, D.; Rossi, C.; Servida, P.; Ugazio, A. G.; Mavilio, F.

Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency, the first genetic disorder treated by gene therapy. Two different retroviral vectors were used to transfer ex vivo the human ADA minigene into bone marrow cells and peripheral blood lymphocytes from two patients undergoing exogenous enzyme replacement therapy. After 2 years of treatment, long-term survival of T and B lymphocytes, marrow cells, and granulocytes expressing the transferred ADA gene was demonstrated and resulted in normalization of the immune repertoire and restoration of cellular and humoral immunity. After discontinuation of treatment, T lymphocytes, derived from transduced peripheral blood lymphocytes, were progressively replaced by marrow-derived T cells in both patients. These results indicate successful gene transfer into long-lasting progenitor cells, producing a functional multilineage progeny.

1995 - Myogenic conversion of mammalian fibroblasts induced by differentiating muscle cells [Articolo su rivista]
Salvatori, G.; Lattanzi, L.; Coletta, R.; Aguanno, S.; Vivarelli, E.; Kelly, R.; Ferrari, G.; Harris, A. J.; Mavilio, F.; Molinaro, M.; Cossu, G.

Somite-derived skeletal myoblasts are supposed to be the sole source of muscle fibre nuclei during pre- and postnatal development, but evidence is accumulating for unorthodox contributions to muscle fibre nuclei from other cell types. For example, in tissue culture, fibroblasts can fuse with dysgenic myoblasts and restore correct membrane function. We report here the results of a series of experiments investigating this phenomenon and its possible mechanism, 10T1/2 cells, infected with a replication defective retrovirus encoding the bacterial enzyme β-galactosidase, fused to form β-galactosidase positive, differentiated myotubes when cocultured with differentiating uninfected C2C12 or primary myogenic cells, but this did not occur when they were cocultured with other cells such as 3T3 fibroblasts or PC12 pheochromocytoma cells. Myogenic conversion ranged from 1 to 10% of the 10T1/2 cell population and required close cell interaction between the different cells types: it was not induced by conditioned medium or extracellular matrix deposited by C2C12 cells. Myogenic conversion was also observed in vivo, after injection of similarly infected 10T1/2 cells into regenerating muscle. Conversion was seen also after coculture of uninfected 10T1/2 cells with primary chick myoblasts, thus demonstrating that it was not dependent upon viral infection and that there is no species or class barrier in this phenomenon. Primary fibroblasts, isolated from different organs of transgenic mice carrying a Lac Z marker under the control of a muscle-specific promoter, restricting β-galactosidase expression to striated muscle cells, also underwent myogenic conversion, when cocultured with C2C12 myoblasts. The efficiency of this conversion varied with their embryological origin, being common in cells with a dorsal mesoderm lineage but rare in cells of ventral mesoderm origin. These experiments demonstrate that myogenic conversion is a true embryological feature of mammalian mesodermal cells. Conversion of mononucleated cells was also observed, showing that fusion is not a pre-requisite for myogenic differentiation and may indeed be a consequence of differentiation induced by short-range local signalling. We conclude that a proportion of adult cells of mesodermal origin may conserve a bi- or multi-potential state of determination throughout the life of an animal, enhancing the regenerative capacity of the tissues in which they reside.

1995 - Retinoic acid induces stage-specific antero-posterior transformation of rostral central nervous system [Articolo su rivista]
Simeone, A.; Avantaggiato, V.; Moroni, M. C.; Mavilio, F.; Arra, C.; Cotelli, F.; Nigro, V.; Acampora, D.

We report a time-course analysis of the effect of retinoic acid (RA) on the development of the mouse central nervous system (CNS) from the beginning of gastrulation throughout induction and patterning of the neural tube. RA administration induces three different, stage-specific alterations of brain development, indicating perturbation of different morphogenetic steps during the establishment of a neural pattern. In particular, treatment at mid-late streak stage (7.2-7.4 days post coitum (d.p.c.)) results in early repression of Otx2 expression in the posterior neuroectoderm of the head fold and in the ventral mid line, including the prechordal plate and the rostralmost endoderm, followed by loss of forebrain morphological and molecular identities, as revealed by analysis of the expression of regionally-restricted brain genes (Otx2, Otx1, Emx2, Emx1 and Dlx1). In these embryos, reduction of the Otx2 expression domain correlates with hindbrain expansion marked by rostral extension of the Hoxb-1 expression domain. Our analysis indicates that RA interferes with the correct definition of both planar and vertical morphogenetic signals at specific developmental stages by affecting gene expression in the regions which are likely either to produce or to respond to these signals. We suggest that retinoids may contribute to early definition of head from trunk structures by selecting different sets of regulatory genes. © 1995.

1995 - Transfer of the HSV-tk Gene into Donor Peripheral Blood Lymphocytes for In Vivo Modulation of Donor Anti Tumor Immunity after Allogeneic Bone Marrow Transplantation. The San Raffaele Hospital, Milan, Italy [Articolo su rivista]
Bordignon, C.; Bonini, C.; Verzeletti, S.; Nobili, N.; Maggioni, D.; Traversari, C.; Giavazzi, R.; Servida, P.; Zappone, E.; Benazzi, E.; Bernardi, M.; Porta, F.; Ferrari, G.; Mavilio, F.; Rossini, S.; Blaese, R. M.; Candotti, F.

The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1–7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor antitumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10,11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1—the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2—the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3—the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes. Patients will be enrolled in three groups: A—patients in complete remission after a T depleted allo-BMT, in order to prevent disease relapse; B—patients with relapse of hematologic malignancies after allo-BMT, in order to achieve a GvL effect; C—patients with an EBV-BLPD after allo-BMT, in order to provide donor EBV-specific T-cells. For this purpose, donor peripheral blood lymphocytes (PBL), will be transduced with a suicide retroviral vector containing two genes: the first coding for the thymidine kinase of the herpes simplex virus (HSV-tk) and the second coding for the low affinity receptor for NGF (12) truncated of its intracellular domain (ΔNGFR). The HSV-tk confers ganciclovir sensitivity (13), thus allowing in vivo selective downregulation of all transduced allogeneic cells in case of severe GvHD. The ΔNGFR will be used as a cell surface marker allowing rapid in vitro immunoselection of transduced cells, and ex vivo detection and characterization of the transduced cells after infusion. © 1995, Mary Ann Liebert, Inc. All rights reserved.

1994 - Inhibition of proliferation and induction of differentiation of pluripotent human embryonal carcinoma cells by osteogenic protein-1 (or bone morphogenetic protein-7) [Articolo su rivista]
Andrews, P. W.; Damjanov, I.; Berends, J.; Kumpf, S.; Zappavigna, V.; Mavilio, F.; Sampath, K.

BACKGROUND: Osteogenic protein-1 (OP-1) is a member of the transforming growth factor-β super family closely related to the bone morphogenetic proteins and also known as bone morphogenetic protein-7. Other members of this family of growth factors influence cell differentiation as well as cell growth in a number of systems. The Drosophila homolog encoded by the decapentaplegic locus is involved in dorsal-ventral pattern formation during embryogenesis, whereas the expression of several bone morphogenetic proteins including OP-1 is developmentally regulated in mammalian embryos. EXPERIMENTAL DESIGN: The effect of recombinant human OP-1 on the proliferation and differentiation of an established pluripotent human embryonal carcinoma (EC) cell line, NTERA2, and three nullipotent human EC cell lines, 2102Ep, 833KE and TERA-1, was investigated. These cells were grown under reduced serum conditions, and differentiation was monitored by morphology and expression of marker antigens. RESULTS: OP-1 inhibited proliferation of NTERA2 and induced their differentiation, marked by changes in cellular morphology, the loss of EC cell antigens (SSEA3, SSEA4, the liver isozyme of alkaline phosphatase), and the appearance of new antigens, notably SSEA1 and class 1 major histocompatibility complex antigens. These changes were irreversible and did not involve significant cell degeneration or cell death. The OP-1-induced differentiation of NTERA2 appeared distinct from that induced by either retinoic acid or hexamethylene bisacetamide. Nevertheless, OP-1 did induce the homeobox gene, HOXA1. By contrast, OP-1 elicited only a limited and partial response from the nullipotent EC cell lines. CONCLUSIONS: Our results suggest that pluripotent human EC cells differentiate in response to OP-1 and that this factor can modulate the differentiation induced by retinoic acid. Like other members of the transforming growth factor-β super family, OP-1 might play an inductive role in the early embryo. The results also suggest a possible therapeutic value for OP-1 in the treatment of some germ cell tumors.

1994 - Inhibition of retinoic acid-induced activation of 3' human HOXB genes by antisense oligonucleotides affects sequential activation of genes located upstream in the four HOX clusters [Articolo su rivista]
Faiella, A.; Zappavigna, V.; Mavilio, F.; Boncinelli, E.

Most homeobox genes belonging to the Hox family are sequentially activated in embryonal carcinoma cells upon treatment with retinoic acid. Genes located at the 3' end of each one of the four Hox clusters are activated progressively later. This activation has been extensively studied for human HOX genes in the NT2/D1 cell line and shown to take place at the transcriptional level. To understand the molecular mechanisms of sequential HOX gene activation in these cells, we tried to modulate the expression of 3' HOX genes through the use of antisense oligonucleotides added to the culture medium. We chose the HOXB locus. A 5- to 15-fold reduction of the expression of HOXB1 and HOXB3 was sufficient to produce a significant inhibition of the activation of the upstream HOXB genes, as well as of their paralogs in the HOXA, HOXC, and HOXD clusters. Conversely, no effect was detectable on downstream HOX genes. The extent of this inhibition increased for progressively more-5' genes. The stability of the corresponding mRNAs appeared to be unaffected, supporting the idea that the observed effect might be mediated at the transcriptional level. These data suggest a cascade model of progressive activation of Hox genes, with a 3'-to-5' polarity.

1994 - Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer [Articolo su rivista]
Mavilio, F.; Ferrari, G.; Rossini, S.; Nobili, N.; Bonini, C.; Casorati, G.; Traversari, C.; Bordignon, C.

Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low- affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cells lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T-cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

1994 - Specificity of HOX protein function depends on DNA-protein and protein- protein interactions, both mediated by the homeo domain [Articolo su rivista]
Zappavigna, V.; Sartori, D.; Mavilio, F.

Transcription of human HOX gene promoters in cultured cells is positively and negatively regulated by HOX proteins interacting with specific target sequences. The human HOXD9 protein activates transcription of the HOXD9 promoter by interacting with the HCR sequence and is antagonized by the HOXD8 protein. HOXD8 is not intrinsically a repressor, since it can activate transcription on different targets. Complete or partial HOXD8/HOXD9 homeo domain swapping indicates that the ability to recognize, and activate transcription from, the HCR target in vivo depends on the amino terminus and helix 1 of the homeo domain. The inhibitory activity of HOXD8 is not affected by deletion of the homeo domain helix 2/3 region, whereas it requires the amino terminus/helix 1 region and an additional, effector domain located at the protein amino-terminal end. This activity is therefore DNA-binding independent, and possibly mediated by protein-protein interactions. Affinity chromatography experiments show that the homeo domain amino terminus/helix 1 region is able to mediate direct interactions between HOX proteins in solution. These data indicate that specificity of HOX protein function in vivo depends on both DNA-protein and protein-protein interactions, mediated by the same sub region of the homeo domain.

1994 - The thyroid transcription factor-1 gene is a candidate target for regulation by Hox proteins [Articolo su rivista]
Guazzi, S.; Lonigro, R.; Pintonello, L.; Boncinelli, E.; Di Lauro, R.; Mavilio, F.

Vertebrate Hox homeobox genes are transcription factors which regulate antero-posterior axial identity in embryogenesis, presumably through activation and/or repression of downstream target genes. Some of these targets were reported to code for molecules involved in cell-cell interactions, whereas no relationship has yet been demonstrated between Hox genes and other transcription factors involved in determining and/or maintaining tissue specificity. The thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein required for expression of thyroid-specific genes. A 862 bp 5' genomic fragment of the rat TTF-1 gene, conferring thyroid-specific expression to a reporter gene, was sufficient to mediate transactivation by the human HOXB3 gene in co-transfection assay in both NIH3T3 or HeLa cells. HOXB3 is expressed in early mammalian embryogenesis in the anterior neuroectoderm, branchial arches and their derivatives, including the area of the thyroid primordia and thyroid gland. Transcription of the TTF-1 promoter is induced only by HOXB3, while its paralogous gene HOXD3 or other Hox genes expressed more posteriorly (HOXA4, HOXD4, HOXC5, HOXC6, HOXC8 and Hoxd-8) have no effect. Transactivation by HOXB3 is mediated by two binding sites containing an ATTA core located at -100 and +30 from the transcription start site. DNase I footprinting experiments show that the two sites bind HOXB3 protein synthesized in both Escherichia coli and eukaryotic cells, as well as nuclear factor(s) present in protein extracts obtained from mouse embryonic tissues which express group 3 Hox genes and TTF-1. Some of the DNA-protein complexes formed by the embryonic extracts are indistinguishable from those generated by HOXB3. These data suggest that HOXB3 might be a transcriptional regulator of the TTF-1 gene in early embryogenesis, and could therefore participate in the specification and development of the thyroid gland.

1993 - Gene therapy [Articolo su rivista]
Mavilio, F.; Bordignon, C.

1993 - Regulation of the human HOXD4 gene by retinoids [Articolo su rivista]
Moroni, M. C.; Vigano, M. A.; Mavilio, F.

Hox genes are developmentally regulated in mammalian embryogenetics, according to temporally and spatially restricted patterns which are affected by retinoids, vitamin A derivatives which have a function as, or at least mimic the action of, axis-specifying morphogens. In the human embryonal carcinoma cell line NT2/D1, HOX gene clusters are activated by at least two retinoids, all-trans- and 9-cis-retinoic acid (RA), in a 3′ to 5′ sequential cascade which reproduces the activation pattern observed in early embryogenesis. We have studied the regulation of the early activated HOXD4 gene, which is expressed in human embryogenesis in multiple transcripts generated by the developmentally controlled use of alternative transcription start sites and polyadenylation sites. Transfection of a 2.9 kb HOXD4 upstream genomic region linked to a reporter gene in NT2/D1 cells, allowed the identification of two different promoters and a distal enhancer element necessary for RA-dependent gene activation. This element confers to a heterologous promoter the ability to be induced by RA in NT2/D1 cells, and transactivated by α, β and γ retinoic acid receptors (RARs), but not retinoid X receptor (RXR), in COS-7 cells. DNase I footprinting analysis allowed the identification of four sequences which bind nuclear factors from both RA-induced NT2/D1 cells and embryonic tissues with similar patterns. The use of specific antibodies allowed the identification of at least RARß in some of the DNA-protein complexes, although the four sequences bind single RARs transfected in COS cells much less efficiently, or not at all, when compared to a canonical RAR responsive element. Induction of the HOXD4 promoter-enhancer in the presence of a selective RARα antagonist indicated that the RARα-dependent RARß activation is nevertheless a necessary step in HOX gene activation. Our results indicate that the ligand-dependent activity of RARs upon specific, cis-acting regulatory elements may have a key role in the induction of early activated HOX genes in response to retinoids. However, RARs repressent only a fraction of the transcription factors interacting with the RA-responsive HOXD4 enhancer, which appears to be a complex element requiring specific combinations of nuclear factors for its proper function. © 1993.

1993 - Regulation of vertebrate homeobox‐containing genes by morphogens [Articolo su rivista]
Mavilio, F.

1993 - Retroviral vector-mediated gene transfer into human primary myogenic cells leads to expression in muscle fibers in vivo [Articolo su rivista]
Salvatori, G.; Ferrari, G.; Mezzogiorno, A.; Servidei, S.; Coletta, M.; Tonali, P.; Giavazzi, R.; Cossu, G.; Mavilio, F.

Primary human myogenic cells isolated from fetal and adult muscle were infected with a high-titer, Moloney murine leukemia virus (MoMLV)-derived retroviral vector expressing a bacterial β-galactosidase (β gal) gene under long terminal repeat (LTR) control. Gene transfer efficiency averaged 50% in both fetal myoblasts and adult satellite cells, as revealed by β-gal staining. The reporter gene was stably integrated, faithfully inherited. and expressed at significant levels in myogenic cells for at least 10 generations under clonal growth conditions, and throughout the culture life span upon differentiation into myotubes. Comparable gene transfer efficiency was obtained in myogenic cells from muscle biopsies of patients affected by a number of genetic or acquired myopathies, including Duchenne muscular dystrophy. Transduced normal human satellite cells were injected into regenerating muscle of immunodeficient mice, where they formed new muscle fibers in which the product of the reporter gene was detectable for 2 months after injection. These results show that retroviral vectors can be used to transfer foreign genes with high efficiency into normal or abnormal primary human myogenic cells, leading to stable expression into mature muscle. Satellite cells engineered in this way might represent an effective tool for gene therapy of muscular dystrophies as well as for systemic delivery of recombinant gene products for correction of inherited and acquired disorders. The human-mouse model described here will allow in vivo testing of such gene therapy approaches.

1992 - The upstream region of the human homeobox gene HOX3D is a target for regulation by retinoic acid and HOX homeoproteins [Articolo su rivista]
Arcioni, L.; Simeone, A.; Guazzi, S.; Zappavigna, V.; Boncinelli, E.; Mavilio, F.

We studied the structure, regulation and expression of HOX3D, a human homeobox gene located in the HOX3 cluster on chromosome 12. HOX3D is developmentally regulated during embryogenesis and is activated by retinoic acid (RA) in cultured embryonal carcinoma (EC) cells. Transfection of HOX3D upstream genomic sequences linked to a reporter gene allowed the functional definition of its promoter, containing a canonical TATA element. This promoter directs the expression of the reporter gene in EC cells after induction with RA, and binds RA-induced nuclear factor(s) through a conserved palindromic sequence located ~ 100 bp upstream of the transcription start site. The HOX3D promoter is transactivated in both human and murine cells when cotransfected with vectors expressing the protein product of the upstream gene HOX3C and the paralogs of further upstream genes in the HOX4 cluster (i.e. HOX4D, HOX4C and the murine HOX 4.3). The HOX3D protein, and those encoded by the downstream gene HOX3E and its paralog HOX4B are instead inactive. HOX4C and HOX4D proteins synthesized in bacteria bind to the same conserved sequence located around position -120, as well as to the TATA box and immediately upstream and downstream nucleotides. These data provide evidence that cross-regulatory interactions between mammalian homeogenes take place in cultured cells, thus raising the possibility that a regulatory network may exist in vivo. The sequences on the HOX3D promoter involved in cross-regulation are different from those binding nuclear factors induced by RA.

1992 - Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions [Articolo su rivista]
Ferrari, G.; Rossini, S.; Nobili, N.; Maggioni, D.; Garofalo, A.; Giavazzi, R.; Mavilio, F.; Bordignon, C.

Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) β gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long- term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4 phenotype. Retroviral integrations and TCR-β gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA- deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.

1991 - An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency [Articolo su rivista]
Ferrari, G.; Rossini, S.; Giavazzi, R.; Maggioni, D.; Nobili, N.; Soldati, M.; Ungers, G.; Mavilio, F.; Gilboa, E.; Bordignon, C.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency (SCID), a candidate genetic disorder for somatic cell gene therapy. Peripheral blood lymphocytes from patients affected by ADA- SCID were transduced with a retroviral vector for human ADA and injected into immunodeficient mice. Long-term survival of vector-transduced human cells was demonstrated in recipient animals. Expression of vector-derived ADA restored immune functions, as indicated by the presence in reconstituted animals of human immunoglobulin and antigen-specific T cells. Retroviral vector gene transfer, therefore, is necessary and sufficient for development of specific immune functions in vivo and has therapeutic potential to correct this lethal immunodeficiency.

1991 - Characterization of Two Neuroblastoma Cell Lines Expressing Recombinant Nerve Growth Factor Receptors [Articolo su rivista]
Reddy, U. R.; Venkatakrishnan, G.; Roy, A. K.; Chen, J.; Hardy, M.; Mavilio, F.; Rovera, G.; Pleasure, D.; Ross, A. H.

Abstract: In earlier studies, a 75,000‐dalton glycoprotein (gp75) has been identified as a component of both low‐ and high‐affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF‐induced up‐regulation of fos oncogene expression, and did not efficiently internalize NGF. LANS is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high‐affinity NGFRs but no detectable low‐affinity sites. The transformant line LAN5/NGFR expressed both high‐ and low‐affinity NGFRs, displayed NGF‐induced up‐regulation of fos oncogene, and efficiently internalized NGF. The number of high‐affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high‐affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high‐affinity NGFRs, the putative second factor may also limit NGF responsiveness. Copyright © 1991, Wiley Blackwell. All rights reserved

1991 - Differential activation of homeobox genes by retinoic acid in human embryonal carcinoma cells [Capitolo/Saggio]
Bottero, L.; Simeone, A.; Arcioni, L.; Acampora, D.; Andrews, P. W.; Boncinelli, E.; Mavilio, F.

1991 - Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells [Articolo su rivista]
Simeone, A.; Acampora, D.; Nigro, V.; Faiella, A.; D'Esposito, M.; Stornaiuolo, A.; Mavilio, F.; Boncinelli, E.

We studied the expression of 38 human homeobox genes belonging to the four HOX complex loci in embryonal carcinoma (EC) cells induced to differentiate by culturing them in a medium containing retinoic acid (RA). Genes located at the 3′ end of each one of the four HOX loci are activated by RA in a sequential order colinear with their 3′ to 5′ arrangement in the cluster: 3′ HOX genes respond early to the drug while upstream genes respond progressively later. Among the genes located at the 5′ end of HOX loci RNase protection analysis reveals that one HOX3 gene and four HOX4 genes are weakly expressed in EC stem cells and downregulated upon treatment with 10-5 M RA. While activation of early responding genes does not require continuous protein synthesis, the observed timing and polarity of gene activation is disrupted in the absence of protein synthesis. © 1991.

1991 - Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma in vivo [Articolo su rivista]
Colombo, M. P.; Ferrari, G.; Stoppacciaro, A.; Parenza, M.; Rodolfo, M.; Mavilio, F.; Parmiani, G.

We have investigated the effect of granulocyte colony-stimulating factor (G-CSF) delivery at the site of tumor growth by transducing, via retroviral vector, the human (hu) G-CSF gene into the colon adenocarcinoma C-26 and assaying the ability of transduced cells to form tumors when injected into syngeneic mice. As a control, the same tumor cells were infected with retroviruses engineered to transduce an unrelated gene, the human nerve growth factor receptor, or carry the neomycin resistance gene only. Only cells transduced with the huG-CSF were unable to develop tumors, although huG-CSF was expressed and produced at low level as estimated by both RNA analysis and enzyme-linked immunosorbent assay, indicating that G-CSF can exert an antitumor effect at a physiological dose. Implication of G-CSF as mediator of tumor inhibition was proven by reversing the nontumorigenic phenotype of G-CSF-expressing cells with anti-huG-CSF monoclonal antibody injected at the tumor site. No tumors were formed by injecting C-26 infected cells into nu/nu mice, while neoplastic nodules appeared after injection into sublethally irradiated mice; such tumors, however, regressed when mice normalized their leukocyte counts after irradiation. Tumors were also formed after injection of a mixture of infected and uninfected C-26 cells, although critical delay in tumor formation occurred when infected cells were 10 times more represented in the mixture. Histological examination of tissues surrounding the site of injection showed infiltration of neutrophilic granulocytes, whose number correlated with that of G-CSF-expressing C-26 cells in the injected mixture. These results indicate that G-CSF may have a potent antitumoral activity when released, even at low doses, at the tumor site. The antitumoral effect is mediated by recruitment and targeting of neutrophilic granulocytes to G-CSF-releasing cells.

1991 - HOX gene activation by retinoic acid [Articolo su rivista]
Boncinelli, E.; Simeone, A.; Acampora, D.; Mavilio, F.

Vertebrate homeobox genes of the Hox family are, like Drosophila homeotic genes, organized in gene clusters and show a strict correspondence, or collinearity, between the order of the genes (3' to 5') within the chromosomal cluster and that of their expression domains (anterior to posterior) in the embryo. Recent data obtained from embryonal carcinoma cells induced to differentiate by retinoic acid cast some light on the molecular mechanisms underlying the collinear expression of the Hox genes.

1990 - Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells [Articolo su rivista]
Simeone, A.; Acampora, D.; Arcioni, L.; Andrews, P. W.; Boncinelli, E.; Mavilio, F.

RETINOIC acid has been implicated as a natural morphogen in chicken1-4 and frog5 embryogenesis, and is presumed to act through the gene regulatory activity of a family of nuclear receptors6-10. Homeobox genes, which specify positional information in Drosophila and possibly in vertebrate embryogenesis11-21, are among the candidate responsive genes4,5,14. We previously reported that retinoic acid specifically induces human homeobox gene (HOX) expression in the embryonal carcinoma cell line NT2/D1 (ref. 22). We now show that the nine genes of the HOX2 cluster are differentially activated in NT2/D1 cells exposed to retinoic acid concentrations ranging from 10-8 to 10-5 M. Genes located in the 3' half of the cluster are induced at peak levels by 10-8 M retinoic acid, whereas a concentration of 10-6 to 10-5 M is required to fully activate 5' genes. At both high and low retinoic acid concentrations, HOX2 genes are sequentially activated in embryonal carcinoma cells in the 3' to 5' direction. © 1990 Nature Publishing Group.

1989 - Alteration of growth and differentiation factors response by Kirsten and Harvey sarcoma viruses in the IL-3-dependent murine hematopoietic cell line 32D C13(G) [Articolo su rivista]
Mavilio, F.; Kreider, B. L.; Valtieri, M.; Naso, G.; Shirsat, N.; Venturelli, D.; Reddy, E. P.; Rovera, G.

32D C13(G) is an interleukin 3(IL3)-dependent nontumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocytic colony stimulating factor (G-CSF). Infections of 32D C13(G) cells with either Kirsten rat sarcoma virus or Balb murine sarcoma virus, both containing a v-ras oncogene, generates clones that can permanently grow in G-CSF without differentiation. 32D-Ki-ras cells show a heterogeneous morphology ranging from the promyelocytic to the myelocytic stage of differentiation, and express high levels of both myeloperoxidase (MPO) and lactoferrin (LF) mRNA. 32D-Ha-ras cells show a more immature phenotype and express MPO but no LF mRNA. The apparent differentiation block of both 32D Ki-ras and 32D Ha ras can be reversed by treatment with the chemical inducers retinoic acid, sodium butyrate or dimethylsulphoxide, which leads to terminal differentiation into granulocytes. When 32D-Ki-ras and 32D-Ha-ras cells are cultured in medium containing IL-3 they become adherent and express some monocyte-macrophage markers. Upon prolonged exposure to IL3, 32D-Ki-ras, but not 32D-Ha-ras, resume suspension growth. Both 32D-Ki-ras and 32D-Ha-ras rapidly die if grown in chemically defined medium in the absence of any growth factor and are non-tumorigenic in immunosuppressed mice. These findings indicate that ras activation may interfere with the normal response to growth and differentiation factors in cells of the granulocytic lineage. These alterations may represent a critical, although non-sufficient, step in leukemogenesis.

1989 - Alteration of the Program of Terminal Differentiation Caused by Oncogenes in the Hemopoietic Progenitor Cell Line 32D C13 (G) [Articolo su rivista]
Rovera, G.; Kreider, B.; Shirsat, N.; Venturelli, D.; Naso, G.; Mavilio, F.

1989 - Constitutive Expression and Abnormal Glycosylation of Transferrin Receptor in Acute T-Cell Leukemia [Articolo su rivista]
Petrini, M.; Pelosi-Testj, E.; Sposi, N. M.; Mastroberardino, G.; Camagna, A.; Bottero, L.; Mavilio, F.; Testa, U.; Peschle, C.

The expression of transferrin receptors (TrfRs) was investigated in acute T-cell leukemia (T-ALL) blasts at the molecular, biochemical, immunological, and functional level. TrfRs, although not detected on quiescent T-cells from normal adults, are constitutively expressed at high level on the blasts from all T-ALL patients and bind normally to transferrin. Their number is modulated by the intracellular iron level, but is independent of exogenous interleukin 2. They also exhibit immunological and biochemical abnormalities, in that: (a) they react preferentially with monoclonal antibodies (MAb) that recognize ligand-binding domains of TrfR (42/6 and 43/31), as compared to MAbs (B3/25, OKT9) that interact with the nonligand binding domains; (b) they have a reduced molecular weight, as compared to TrfR on normal thymocytes and activated T-lymphocytes: this phenomenon is apparently related to a defective glycosylation. It is noteworthy that expression of TrfR was not observed in a large series of other types of acute leukemias, i.e., pre-B, B, and myeloid leukemias, excluding erythroleukemias. The constitutive, high level expression of TrfRs on T-ALL blasts may play a key role in the stepwise progression of this malignancy and particularly provide a proliferative advantage to T-ALL blasts as compared to normal T-lymphocytes. Furthermore, indirect evidence suggests that the glycosylation defect of TrfR on T-ALL blasts contributes to their tumorigenic capacity. © 1989, American Association for Cancer Research. All rights reserved.

1989 - Posttranscriptional control of human homeobox gene expression in induced NTERA‐2 embryonal carcinoma cells [Articolo su rivista]
Simeone, A.; Acampora, D.; D'Esposito, M.; Faiella, A.; Pannese, M.; Scotto, L.; Montanucci, M.; D'Alessandro, G.; Mavilio, F.; Boncinelli, E.

We have studied the expression of four human homeobox genes representative of four different clusters (i.e., HOX‐1, HOX‐2, HOX‐3 and HOX‐5) in the embryonal carcinoma (EC) cell line NT2/D1. Following treatment with retinoic acid (RA), these cells differentiate into several cell types, including neurons, and steadily accumulate polyadenylated transcripts derived from the genes in a period ranging from 18 hr to 14 days of RA treatment. The sizes of major transcripts in differentiated EC cells coincide with those previously detected by the same probes in human embryos. Nuclear run‐on transcriptional analysis showed no difference in the transcription rate of the four homeobox genes in differentiated vs. undifferentiated EC cells. Inhibition of protein synthesis by 5–18 hr of treatment of undifferentiated cells with cycloeximide causes accumulation of some homeobox transcripts at levels comparable to those observed after 18 hr of RA induction, although it does not cause superinduction in fully differentiated cells. These data suggest that the activation of homeobox gene expression in RA‐induced EC cells is controlled, at least in part, by posttranscriptional mechanisms. Copyright © 1988 Wiley‐Liss, Inc.

1988 - Activation of four homeobox gene clusters in human embryonal carcinoma cells induced to differentiate by retinoic acid [Articolo su rivista]
Mavilio, F.; Simeone, A.; Boncinelli, E.; Andrews, P. W.

We have studied the expression of nine homeobox genes from Hox I, Hox 2, Hox 3 and Hox 5 dusters in human embryonal carcinoma (EC) eel! lines analyzed as both stem cells and after exposure to the differentiation-inducing agents retinoic acid (RA), hcxamethylencbisacetamide (HMBA)and bromodeoxyuridine (BUdR). None of the homeobox genes was expressed in stem cells, whereas all were activated, although with different kinetics, in cultures of the pluripotent EC cell line NTERA-2, clone Dl (NT2/ Dl), following differentiation induced by RA. At least some bomeobox genes were stably expressed in differentiated cells several weeks after removal of RA from the culture medium. However, the length of initial exposure to RA is a critical factor in achieving stable gene expression, and differs among the different sets of genes and, at least in one case, among different transcripts from the same gene. No homeobox gene expression was detected in NT2/D1 cells induced to differentiate with HMBA or BUdR. Also, no expression was detectable in xenograft tumors generated by NT2/D1 cells in nude mice, even though tumors of this type contain mostly differentiated cells. Other human EC lines tested, i.e., 833 KE, 2102Ep ur 1156QE, did not differentiate in response to RA and did not express homeobox genes. No expression was detectable in xenograft tumors of 833KE and 2I02Ep, containing essentially EC cells. These data indicate that homeobox-gene activation specifically accompanies RA-induced differentiation of NT2/D1 cells, thereby providing an excellent model for studying the molecular basis of homeobox-gene regulation and the possible role of the homeobox in cell differentiation. © 1988, International Society of Differentiation. All rights reserved.

1987 - Cytokine-dependent granulocytic differentiation: Regulation of proliferative and differentiative responses in a murine progenitor cell line [Articolo su rivista]
Valtieri, M.; Tweardy, D. J.; Caracciolo, D.; Johnson, K.; Mavilio, F.; Altmann, S.; Santoli, D.; Rovera, G.

Human granulocyte colony stimulating factor (G-CSF) can support the survival and short term proliferation of the interleukin 3 (IL 3)-dependent diploid murine hemopoietic progenitor cell line 32D C13. After 8 days in the presence of 30 U/ml of G-CSF and in the absence of IL 3, the great majority of 32D C13 cells becomes positive for myeloperoxidase (a marker that appears at the promyelocytic stage of the granulocytic lineage) and progressively differentiates into lactoferrin-containing neutrophilic granulocytes. Myeloperoxidase mRNA rapidly increases after 24 to 48 hr of treatment with G-CSF, peaks at day 6 and is no longer detectable at day 9 and 12, paralleling the appearance of myeloperoxidase-positive promyelocytes and myelocytes in the culture. After 12 days, 100% of the cells terminally differentiate, and clonogenic assays in IL 3-containing semisolid media indicate that the whole population has irreversibly lost proliferative capability. By using varying concentrations of both murine IL 3 and recombinant human G-CSF, the cultures develop an heterogeneous population of cells representing all the differentiation stages of the myeloid lineage, and the relative ratios of immature proliferating precursors and terminally differentiated cells present in the cultures can be modulated by modifying the concentrations of IL 3 or recombinant human G-CSF. Isobolic curves indicate that IL 3 and G-CSF have an antagonistic effect on the proliferation of 32D C13 cells. Thus, these cells represent a simplified in vitro model of normal granulocytic differentiation whose extent may be modulated completely in the presence of serum by two well-defined growth and differentiation factors: IL 3 and G-CSF.

1987 - Differential regulation of transferrin receptor gene expression in human hemopoietic cells: Molecular and cellular aspects [Articolo su rivista]
Testa, U.; Testa, E. P.; Mavilio, F.; Petrini, M.; Sposi, N. M.; Petti, S.; Samoggia, P.; Montesoro, E.; Giannella, G.; Bottero, L.; Camagna, A.; Salvo, G.; Isacchi, G.; Habetswallner, D.; Peschle, C.

1987 - Effect of Abelson murine leukemia virus on granulocytic differentiation and interleukin-3 dependence of a murine progenitor cell line [Articolo su rivista]
Rovera, G.; Valtieri, M.; Mavilio, F.; Premkumar Reddy, E.

The murine diploid hematopoietic cell line 32D C13 strictly requires interleukin-3 (IL-3) for proliferation. When 32D C13 cells are transferred to IL-3-free medium which contains recombinant human granulocyte colony stimulating factor (rhG-CSF), the cell number increases four- to five-fold, and after 14 days the whole cell population is differentiated into morphologically normal and myeloperoxidase- and lactoferrin-positive metamyelocytes and granulocytes. Infection with Abelson murine leukemia virus (A-MuLV) of 32D C13 cells growing in the presence of IL-3 induces, within 2 weeks, the appearance of cells that are IL-3-independent for growth. The latter cells lack myeloid, T and B cell markers, and are unable to differentiate, even in the presence of very high doses of rhG-CSF. However, once the 32D C13 cells have been exposed to G-CSF, they become resistant to the transforming effects of A-MuLV as judged by the appearance of the IL-3-independent clones. These findings suggest that the ability of Abelson virus to transform immature progenitor cells is due to interference of the v-abl gene product with the mechanism that control the commitment of the cells to differentiate.

1987 - Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulatory factor for growth [Articolo su rivista]
Valtieri, M.; Santoli, D.; Caracciolo, D.; Kreider, B. L.; Altmann, S. W.; Tweardy, D. J.; Gemperlein, I.; Mavilio, F.; Lange, B.; Rovera, G.

A human leukemia cell line (TALL-101) was established from the bone marrow of a patient with an undifferentiated acute T cell leukemia using the conditioned medium (CM) of the human T cell leukemia virus (HTLV) II-transformed human cell line J-LB1. Immunofluorescence analysis on the original leukemic cells indicated the presence of T cell markers (Leu-1, Tdt, and T11); however, the established TALL-101 cell line expressed only antigens commonly present on progenitor cells, thymocytes, and myelomonocytic cells, but not on mature T cells. A high percentage of TALL-101 cells displayed the Tac antigen which was down-regulated upon incubation in the presence of recombinant human (rH) interleukin 2 (IL 2). Interferon (IFN)-γ induced the appearance of class II histocompatibility leukocyte antigens (HLA) and of a T cell marker (3A1), and enhanced the expression of transferrin receptors on these cells. Further evidence for a T cell lineage of the TALL-101 cell line was provided by both chromosomic and genotypic analysis showing a translocation in chromosome 14 typical of T cell leukemias, and a rearrangement of the T-β receptor locus. The growth-promoting activity in the J-LB1-CM was identified as granulocyte-macrophage colony stimulatory factor (GM-CSF), a growth factor which stimulates proliferation of normal myelomonocytic cells and other progenitor cells, but not known to have an effect on T cells. Dose response curves of [3H]thymidine incorporation and growth indicated that TALL-101 cells were sensitive to very low concentrations of rHGM-CSF, 5 ng/ml inducing maximal proliferation in chemically defined medium. The TALL-101 cell line is strictly GM-CSF-dependent for growth: upon depletion of GM-CSF from the culture medium, the cells stop proliferating immediately and die within 1 to 2 wk. The overall data, showing that GM-CSF is able to support the growth of a highly undifferentiated T cell leukemia, strongly suggests that this factor might have similar growth promoting effects on other immature T cell leukemias, and possibly, on normal T cell progenitors.

1987 - Expression of Transferrin Receptors: Differential Regulatory Mechanisms in Monocytes‐macrophages versus Other Hemopoietic Cells [Articolo su rivista]
Testa, U.; Camagna, A.; Giannella, G.; PELOSI-TESTA, E.; Petrini, M.; Samoggia, P.; Montesoro, E.; Bottero, L.; Sposi, N. M.; Salvo, G.; Mavilio, F.; Isacchi, G.; Mastroberardino, G.; Peschle, C.

1987 - Expression of c‐fos in Human Normal and Neoplastic Monocyte‐Macrophage Differentiation [Articolo su rivista]
Sposi, N. M.; Petrini, M.; Mavilio, F.; Testa, U.; Bottero, L.; Pelosi, E.; Mastroberardino, G.; Amadori, S.; Mandelli, F.; Peschle, C.

1987 - Growth factor requirements of childhood acute leukemia: Establishment of GM-CSF-dependent cell lines [Articolo su rivista]
Lange, B.; Valtieri, M.; Santoli, D.; Caracciolo, D.; Mavilio, F.; Gemperlein, I.; Griffin, C.; Emanuel, B.; Finan, J.; Nowell, P.

Eight permanent cell lines were established from cells of 50 consecutive patients with childhood acute leukemia. Three cell lines required growth factor-containing conditioned media. Analysis using blocking antisera and recombinant granulocytic macrophage (GM) colony-stimulating factor (CSF) identified GM-CSF as a growth factor required to establish the latter three cell lines and necessary for their continuous proliferation in chemically defined medium. Two of the GM-CSF-dependent cell lines were derived from patients with undifferentiated T- and a biphenotypic B-myelomonocytic leukemia, which suggests that GM-CSF might maintain proliferation of leukemias originating from immature progenitor cells. Cytogenetic analysis indicated that all established leukemic cell lines were aneuploid, with six lines containing chromosomal alterations related to those observed in the leukemic cells of the patient. Two patients did not have an abnormal clone identified in the marrow but did yield an aneuploid cell line. These studies indicate that GM-CSF-dependent leukemic cell lines can be established in a fraction of childhood leukemia. These cell lines lend themselves to studies aimed at the evaluation in vitro of the role of growth factors in controlling proliferation and differentiation of leukemic cells.

1987 - Human acute myelogenous and lymphoid leukemias: pattern of expression of cellular oncogenes [Articolo su rivista]
Sposi, N. M.; Mavilio, F.; Petrini, M.; Bottero, L.; Zappavigna, V.; Mastroberardino, G.; De Rossi, G.; Amadori, S.; Mandelli, F.; Peschle, C.

1987 - Selective expression of fos proto-oncogene in human acute myelomonocytic and monocytic leukemias: A molecular marker of terminal differentiation [Articolo su rivista]
Mavilio, F.; Testa, U.; Sposi, N. M.; Petrini, M.; Pelosi, E.; Bordignon, C.; Amadori, S.; Mandelli, F.; Peschle, C.

Expression of human fos proto-oncogene (c-fos) was analyzed in primary cells from 50 untreated acute lymphocytic (ALL) and myeloblastic (AML) leukemias. c-fos RNA, analyzed by blot hybridization, was detected virtually only in myelomonocytic (M4) and monocytic (M5) AML. Both M4 and M5 samples show a strong positive correlation between the amount of c-fos transcripts and the percentage of leukemic cells expressing surface antigens specific for mature monocytes and macrophages. Normal mature monocytes exhibit a detectable level of c-fos RNA, which is virtually unaltered on activation to macrophage differentiation, but is always below that observed in M4 through M5 monocyticlike cells. These data provide evidence that c-fos expression is linked to terminal monocyte and macrophage differentiation in normal and leukemic hemopoiesis.

1987 - Two human homeobox genes, c1 and c8: Structure analysis and expression in embryonic development [Articolo su rivista]
Simeone, A.; Mavilio, F.; Acampora, D.; Giampaolo, A.; Faiella, A.; Zappavigna, V.; D'Esposito, M.; Pannese, M.; Russo, G.; Boncinelli, E.

Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomain identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hybridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embros and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny.

1986 - A human homoeo box gene specifically expressed in spinal cord during embryonic development [Articolo su rivista]
Simeone, A.; Mavilio, F.; Bottero, L.; Giampaolo, A.; Russo, G.; Faiella, A.; Boncinelli, E.; Peschle, C.

Several genes involved in the determination of Drosophila body segments share a conserved DNA sequence of 183 nucleotides termed the homoeo box 1. Homologous homoeo box sequences have been detected in the genome of species ranging from insects and anellids to vertebrates2,3, and a number of homoeo box-containing genomic DNA clones have been isolated from Xenopus, mouse and human4-11. We have recently isolated human complementary DNA clones containing homoeo box sequences, representing transcripts from four different genes12. We report here the nucleotide sequence of one of these clones (HHO.c10) and show that the corresponding gene is transcribed in human embryos and fetuses at 5-10 weeks post-conception. A major polyadenylated transcript of ∼2.1 kilobases (kb), as well as RNA species of higher relative molecular mass (Mr), are specifically expressed at a constant level in spinal cord throughout this developmental period. © 1986 Nature Publishing Group.

1986 - Differential and stage-related expression in embryonic tissues of a new human homoeobox gene [Articolo su rivista]
Mavilio, F.; Simeone, A.; Giampaolo, A.; Faiella, A.; Zappavigna, V.; Acampora, D.; Poiana, G.; Russo, G.; Peschle, C.; Boncinelli, E.

The homoeobox is a 183 base-pair (bp) DNA sequence1 conserved in several Drosophila genes controlling segmentation and segment identity 2-4. Homoeobox sequences have been detected in the genome of species ranging from insects and anellids to vertebrates5,6 and homoeobox containing genes have been cloned from Xenopus7-9, mouse 10-22 and man23. We recently isolated human homoeobox containing complementary DNA clones, that represent transcripts from four different human genes24. One clone (HHO.c10) is selectively expressed in a 2.1 kilobase (kb) polyadenylated transcript in the spinal cord of human embryos and fetuses 5-10 weeks after fertilization25. We report the characterization of a second cDNA clone, termed HHO.c13, that represents a new homoeobox gene. This clone encodes a protein of 255 amino-acid residues, which includes a pentapeptide, upstream of the homoeo domain, conserved in other Drosophila, Xenopus, murine and human homoeobox genes. By Northern analysis HHO.c13 detects multiple embryonic transcripts, which are differentially expressed in spinal cord, brain, backbone rudiments, limb buds and heart in 5-9-week-old human embryos and fetuses, in a striking organ- and stage-specific pattern. These observations suggest that in early mammalian development homoeobox genes may exert a wide spectrum of control functions in a variety of organs and body parts, in addition to the spinal cord. © 1986 Nature Publishing Group.

1986 - Expression of cellular oncogenes in primary cells from human acute leukemias [Articolo su rivista]
Mavilio, F.; Sposi, N. M.; Petrini, M.; Bottero, L.; Marinucci, M.; De Rossi, G.; Amadori, S.; Mandelli, F.; Peschle, C.

The structure and the expression of 11 cellular oncogenes (protooncogenes) were analyzed in primary cells from 20 acute lymphocytic (ALL) and 31 acute myelogenous (AML) leukemia patients. Neoplastic cells, obtained prior to initiation of therapy, were purified and classified, on the basis of both surface antigen pattern and morphology, into pre-B, B, and T ALL and M1-M5 AML. RNA was extracted and analyzed for expression of cellular oncogenes coding for nuclear proteins (c-myc, c-myb, c-fos), the β-chain of platelet-derived growth factor (c-sis), factor receptors or related proteins (c-src, c-abl, c-fes, c-erbB), or putative intermediate transducers of mitogenic signals (c-Ha-ras, c-Ki-ras, c-N-ras). Quanatitative analysis of total RNA was carried out by dot blot hybridization to specific cDNA or genomic probes. Number and size of transcripts were evaluated by blot hybridization of electrophoretically fractionated poly(A)+ RNA. Expression of c-myc and c-myb was detected in all leukemic cells at variable levels and was characterized by well-defined patterns within ALL subtypes. Conversely, significant levels of c-fos transcripts were detected only in myelomonocytic (M4) and monocytic (M5) leukemias. Among the 'src-family', c-fes was expressed morein AML than ALL, and c-abl was expressed at variable but not elevated levels in all leukemia types. c-Ha-ras was uniformly expressed at low levels, as in non-neoplastic cells. c-Ki-ras transcription was detected only in T ALL; N-ras expression was barely demonstrable. The structure of these protooncogenes was not grossly modified, as evaluated by Southern analysis, except for c-myc rearrangement in B ALL. These studies indicate that cellular oncogene expression in specific subtypes of leukemic cells may relate to either the proliferative activity (c-myc c-myb) or the differentiation state (c-fos) of the cells, or possibly to expression of receptors for putative hemopoiesis-related growth factors (c-fes, c-abl). Our data provide a basis for in-depth analysis of protooncogene expression in normal and neoplastic hemopoiesis.

1986 - Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac-liver transition [Articolo su rivista]
Migliaccio, G.; Migliaccio, A. R.; Petti, S.; Mavilio, F.; Russo, G.; Lazzaro, D.; Testa, U.; Marinucci, M.; Peschle, C.

Human embryonic development involves transition from yolk sac (YS) to liver (L) hemopoiesis. We report the identification of pluripotent, erythroid, and granulo-macrophage progenitors in YS, L, and blood from human embryos. Furthermore, comprehensive studies are presented on the number of hemopoietic progenitors and precursors, as well as of other cell types, in YS, L, and blood at precisely sequential stages in embryos and early fetuses (i.e., at 4.5-8 wk and 9-10 wk postconception, respectively). Our results provide circumstantial support to a monoclonal hypothesis for human embryonic hemopoiesis, based on migration of stem and early progenitor cells from a generation site (YS) to a colonization site (L) via circulating blood. The YS → L transition is associated with development of the differentiation program in proliferating stem cells: their erythroid progeny shows, therefore, parallel switches of multiple parameters, e.g., morphology (megaloblasts → macrocytes) and globin expression (ζ → α, ε → γ).

1986 - Translocation of c-myc into the immunoglobulin heavy-chain locus in human acute B-cell leukemia. A molecular analysis [Articolo su rivista]
Care, A.; Cianetti, L.; Giampaolo, A.; Sposi, N. M.; Zappavigna, V.; Mavilio, F.; Alimena, G.; Amadori, S.; Mandelli, F.; Peschle, C.

We report the molecular analysis of primary cells from four cases of human B-cell malignancies each with an 8;14 chromosomal translocation involving the c-myc proto-oncogene and the immunoglobulin (Ig) gene cluster. In two cases of B-cell acute lymphocytic leukemia (B-ALL) the c-myc is truncated, rearranged into the Ig C alpha 1 locus and over-expressed in two abnormal mRNAs of approximately 2.0 and 2.8 kb. Conversely, in two cases of B-cell lymphoma progressed into leukemia the c-myc locus was translocated intact in its coding and 5'-flanking region into an Ig region different from C alpha 1, and over-expressed in two normal mRNA species. Cloning and sequencing of the breakpoint region on chromosome 14q+ from one of the two B-ALL cases showed that the myc gene is truncated 1077 nucleotides upstream from the translation start site, and rearranged in the opposite transcriptional orientation into an Ig class-switch segment approximately 4.8 kb upstream from the C alpha 1 gene. The c-myc anti-sense strand contains two class-switch recombination consensus sequences in the immediate boundaries of the breakpoint on chromosome 8: this allows us to postulate that an erroneous, class-switch-like recombination between Ig and myc sequences gave rise to the chromosomal translocation. Furthermore, we report 13 point mutations clustered in a region spanning from the first intron to the second exon of the translocated c-myc gene, five of which cause amino acid changes leading to an abnormal myc protein. This is the first evidence of mutations in a translocated c-myc in primary tumor cells.

1985 - A Model for Hemoglobin F Synthesis in Adult Life: Evidence for Regulation at the Level of Erythroblasts [Articolo su rivista]
Peschle, C.; Migliaccio, A. R.; Migliaccio, G.; Mavilio, F.; Rocca, E.; Petrini, M.; Mastroberardino, G.; Comi, P.; Giglioni, B.; Ottolenghi, S.

1985 - Embryonic hemopoiesis in human liver: morphologic aspects at sequential stages of ontogenic development [Articolo su rivista]
Petti, S.; Testa, U.; Migliaccio, A. R.; Mavilio, F.; Marinucci, M.; Lazzaro, D.; Russo, G.; Mastroberardino, G.; Peschle, C.

1985 - Erythropoietic development and hemoglobin switching in human embryos: cellular and molecular aspects [Articolo su rivista]
Peschle, C.; Mavilio, F.; Migliaccio, G.; Migliaccio, A. R.; Russo, G.; Mastroberardino, G.; Marinucci, M.

1985 - Haemoglobin switching in human embryos: Asynchrony of ζ → α and ε → γ-globin switches in primitive and definitive erythropoietic lineage [Articolo su rivista]
Peschle, C.; Mavilio, F.; Care, A.; Migliaccio, G.; Migliaccio, A. R.; Salvo, G.; Samoggia, P.; Petti, S.; Guerriero, R.; Marinucci, M.; Lazzaro, D.; Russo, G.; Mastroberardino, G.

Haemoglobin switching in humans provides a unique model for investigating the mechanisms underlying expression of a develop-mentally regulated gene family. Numerous studies have focused on the switch from fetal to adult (that is, γ → β) globin1,2, but little is known about the embryonic → fetal (that is, ζ → α and ε → γ) switches, as well as the transition from 'primitive' yolk sac to 'definitive' liver erythropoiesis3-7. Here we have studied the embryonic→fetal haemoglobin switches in yolk sac, liver and circulating blood erythroblasts from 25 embryos and 6 fetuses. Globin synthesis was also evaluated in purified 'primitive' and 'definitive' erythroblasts. Primitive erythroblasts synthesize essentially ζ and ε chains at 5 weeks and α- and ε-globin with a minor aliquot of ζ and γ chains at 6-7 weeks, whereas definitive erythroblasts produce α and γ + β-globin at 6 weeks but only α and γ + β chains from 8 weeks onward. In both lineages the ζ → α and the ε → γ switches are asynchronous, the former preceding the latter. Furthermore, ζ- and β-globin synthesis is restricted to primitive and definitive erythroblasts respectively. These findings are discussed in terms of a monoclonal model for haemoglobin switching in early human ontogeny. © 1985 Nature Publishing Group.

1984 - Association of heterocellular HPFH, β+-thalassaemia, and δβ° -thallassaemia: Haematological and molecular aspects [Articolo su rivista]
Cianetti, L.; Care, A.; Sposi, N. M.; Giampaolo, A.; Calandrini, M.; Petrini, M.; Massa, A.; Marinucci, M.; Mavilio, F.; Ceccanti, M.

An Italian family in which heterocellular hereditary persistence of fetal haemoglobin (HPFH) interacts with both β+- and δβ-thalassaemia is described. The index case was an 8 year old girl who was presumed to inherit both heterocellular HPFH and β+-thalassaemia from her mother and δβ-thalassaemia from her father. She was healthy and never needed blood transfusions. The possible contribution of heterocellular HPFH to the less severe expression of the compound δβ/β+-thalassaemia heterozygosity is discussed. By DNA analysis the specific δβ-thalassaemia defect on the γδβ globin gene region has been established. In addition, a previously unreported association of a polymorphic restriction site haplotype with a β+-thalassaemia mutation has been observed.

1984 - Heterocellular hereditary persistence of fetal hemoglobin (HPFH). Molecular mechanisms of abnormal γ-gene expression in association with β thalassemia and linkage relationship with the β-globin gene cluster [Articolo su rivista]
Giampaolo, A.; Mavilio, F.; Sposi, N. M.; Care, A.; Massa, A.; Cianetti, L.; Petrini, M.; Russo, R.; Cappellini, M. D.; Marinucci, M.

We report a study of four families of Italian origin in which heterocellular HPFH is inherited linked to β thalassemia over two or three generations. The HPFH + β thalassemia carriers showed thalassemic blood pictures and elevated HbF and F-cell number without increase in the HbF/F-cell content. Association of this gene complex with a second β thalassemia trait gives rise to a mild clinical picture characterized by 9-12 g/dl of mainly HbF in peripheral blood and no transfusion requirement. In two families, independent segregation of the HPFH or β-thal trait was observed, and in one case the study of the DNA polymorphisms within the γδβ gene cluster indicated that the HPFH mutation lies outside that DNA region. In one family the coexistence of a polymorphic variant of the Aγ chain (the AγT chain) allowed us to demonstrate that the increased γ chain synthesis caused by the heterocellular HPFH determinant is directed by both chromosomes. © 1984 Springer-Verlag.

1984 - Molecular heterogeneity of beta thalassaemia in the Italian population [Articolo su rivista]
Giampaolo, A.; Mavilio, F.; Massa, A.; Gabbianelli, M.; Guerriero, R.; Sposi, N. M.; Care, A.; Cianciulli, P.; Tentori, L.; Marinucci, M.

Summary Fifty‐one subjects originating from Southern Italy and affected by Cooley's anaemia have been studied in order to define the degree of heterogeneity of β thalassaemia mutations in this high incidence area. Restriction endonuclease mapping has been carried out on genomic DNA by the Southern blot technique both to exclude the existence of gross deletions or rearrangements and to establish the relative frequency of four polymorphic restriction sites (i.e. Gγ and Aγ Hind III, β Ava II and β Bam HI) within the γδβ gene region. In 28 subjects unequivocal linkage of the four polymorphic sites has been determined leading to the identification of seven different chromosome haplotypes, six of which had previously been reported associated with specific β0 and β+ thalassaemia mutations. Globin chain synthesis studies on peripheral blood reticulocytes indicated that subjects carrying the same genotype may behave differently as far as the β chain production is concerned relative to both the a and the non‐α chains. Thus, β thalassaemia turns out to be quite heterogeneous even in this limited geographical area. β+ mutations appear to be predominant, particularly those affecting nuclear precursor RNA splicing to mature β globin mRNA. Copyright © 1984, Wiley Blackwell. All rights reserved

1984 - Translocation and rearrangement of c-myc into immunoglobulin α heavy chain locus in primary cells from acute lymphocytic leukemia [Articolo su rivista]
Peschle, C.; Mavilio, F.; Sposi, N. M.; Giampaolo, A.; Care, A.; Bottero, L.; Bruno, M.; Mastroberardino, G.; Gastaldi, R.; Testa, M. G.

We report the molecular analysis of an 8;14 reciprocal chromsome translocation in a case of acute lymphocytic leukemia (L3 type). DNA from primary leukemic cells was analyzed on the basis of restriction endonuclease mapping by hybridization with various human c-myc and Ig heavy chain probes. The breakpoint of the translocation is within an ~ 200-base-pair region in the first intron of the c-myc gene. The first, untranslated exon thereby remains on chromosome 8q-, whereas the whole protein-coding region is rearranged in the Cα1 locus on chromosome 14q+. RNA transfer blot analysis showed high levels of at least two different c-myc transcripts originated from the translocated gene. Both differ in size from the normal 2.2- and 2.4-kilobase transcripts. Both c-myc structure and expression were apparently normalized in remission phase. These studies demonstrate rearrangement and abnormal expression of c-myc in primary cells from an acute leukemia patient, thus adding to the concept of a key role for c-onc in human oncogenesis.

1983 - Molecular mechanisms of human hemoglobin switching: Selective undermethylation and expression of globin genes in embryonic, fetal, and adult erythroblasts [Abstract in Rivista]
Mavilio, F.; Giampaolo, A.; Care, A.; Migliaccio, G.; Calandrini, M.; Russo, G.; Pagliardi, G. L.; Mastroberardino, G.; Marinucci, M.; Peschle, C.

The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult β-like globin gene domains were evaluated in ≥90% purified human erythroblasts from yolk sacs and fetal livers in the 6- to 12-week gestational period as well as from adult marrows. The 6-wk erythroblasts produce essentially embryonic ε chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult β chains. In all phases of ontogenic development, a strong correlation exists between DNA hypomethylation in the close flanking sequences of globin genes and their expression. These results suggest that modulation of the methylation pattern may represent a key mechanism for regulating expression of human globin genes during embryonic → fetal and fetal → adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-α gene cluster, thus leading to a 5' → 3' activation of globin genes in a balanced fashion.

1983 - The δβ crossover region in Lepore Boston hemoglobinopathy is restricted to a 59 base pairs region around the 5' splice junction of the large globin gene intervening sequence [Articolo su rivista]
Mavilio, F.; Giampaolo, A.; Care, A.; Sposi, N. M.; Marinucci, M.

1982 - β-Thalassemia in southern Italy: A preliminary approach [Articolo su rivista]
Tentori, L.; Marinucci, M.; Massa, A.; Mavilio, F.; Guerriero, R.; Gabbianelli, M.; Giampaolo, A.; Cianciulli, P.; Sposi, N. M.; Masi, M.; Cecconi, M.; Antimi, M.; Pratesi, G.

1981 - Hemoglobin bologna (α2β2 61 (E5) Lys → Met) An abnormal human hemoglobin wlth low oxygen affinity [Articolo su rivista]
Marinucci, M.; Giuliani, A.; Maffi, D.; Massa, A.; Giampaolo, A.; Mavilio, F.; Zannotti, M.; Tentori, L.

An abnormal human hemoglobin was found in association with β-thalassemia in a hemolysate from an 11-year-old healthy child living in Bologna (northern) Italy). Structural studies demonstrated a previously unreported amino acid substitution, β 61 (E5) Lys → Met (this is an external residue). The new variant has been named Hb Bologna, and is characterized by a reduced oxygen affinity. Family studies indicated that the variant had been inherited from the father, a 41-year-old male of Southern Italian origin. Also, a brother of the propositus was found to be an abnormal Hb carrier. © 1981.

1981 - Hemoglobinopathies in Italy. Geographic distribution and criteria for their screening [Articolo su rivista]
Tentori, L.; Marinucci, M.; Massa, A.; Giuliani, A.; Mavilio, F.

1981 - Occurrence of HB m iwate (α2 87 his → tyr β2 in an italian carrier [Articolo su rivista]
Maggio, A.; Massa, A.; Giampaolo, A.; Mavilio, F.; Tentori, L.

1981 - The biosynthesis of hemoglobin G san josé (β 7 (A4) Glu→Gly) [Articolo su rivista]
Marinucci, M.; Giampaolo, A.; Maffi, D.; Mavilio, F.; Cianetti, L.; Mule, F.; Tentori, L.

This report is concerned with the evaluation of hematological parameters and the percentage level of the abnormal hemoglobin (Hb) G San José as found in 4 heterozygous carriers from a family of Sicilian origin. Biosynthetic studies and in vitro recombination experiments strongly indicate that abnormal β chains are synthesized at lower rate than βA chains and exhibit a minor affinity (relative to βA chains) for complementary chains in a condition of relative αA chain deficiency. The possibility that the low affinity of βG chains for α chains may play a decisive role in controlling the level of the abnormal Hb in the peripheral blood of the present non-α-thalassemic abnormal Hb carriers is therefore discussed. © 1981 S. Karger AG, Basel.

1981 - β thalassemia associated with increased HB f production. Evidence for the existence of a heterocellular hereditary persistence of fetal hemoglobin (hpfh) determinant linked to β thalassemia in a southern italian population [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Giuliani, A.; Gabbianelli, M.; Tentori, L.; Tentori, L.; Zorini, C. O.; Lamberti, E.; Palazzolo, A.; Lanzo, D.

A family has been observed in which a β thalassemia determinant is inherited over three generations together with high Hb F level (8-12 % and increased number of fetal-hemoglobin-containing-cells (F-cells). The values of red cell indices and globin chain synthesis ratios, yet typical of β thalassemia, were significantly shifted to the normal values when compared with those of typical β thalassemia heterozygotes belonging to the same family group. The occurrence in these individuals of a heterocellular hereditary persistence of fetal hemoglobin (HPFH) determinant and its linkage relationship with the β thalassemia is discussed. In the third generation two adult individuals were β thalassemia homozygotes having inherited a β thalassemia determinant from one parent and a β thalassemia together with the HPFH determinant from the other. They showed an extremely mild clinical condition, and 11-12 g/dl of mainly Hb F without having ever required blood transfusions. Virtually all the red cells were F-cells in both subjects. The importance of the coexistence of HPFH determinants capable of increasing the size of the F-cell population in patients affected by homozygous thalassemia is discussed, considering the sensible benefit which derives from enhanced Hb F production in this syndrome. © 1981 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1980 - A new human hemoglobin variant: Hb Bari (α2 45 (CD3) His → Gln β2) [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Tentori, L.; D'Erasmo, F.; Colapietro, A.; de Stasio, G.; di Fonzo, S.

An α-chain variant hemoglobin was found in the hemolysate of a 21-year-old healthy male living in Bari (Puglia, Italy). Structural studies demonstrated a previously unreported amino acid substitution, α2 45 (CD3) His → Gln β2, involving a distal heme contact. The new variant has been named Hb Bari. Its electrophoretic behavior was the same as for Hb A; it was stable to both isopropanol and heat denaturation and exhibited normal functional properties, with respect to whole blood and stripped hemolysate studies. The level of Hb Bari was about 20% in the observed carrier. No relative was available for further investigations. © 1980.

1980 - Hemoglobin hasharon [α2 47 (CD5) Asp→His β2] linked to α-thalassemia in northern italian carriers: Hematological and biosynthetic studies [Articolo su rivista]
Mavilio, F.; Marinucci, M.; Massa, A.; Fontanarosa, P. P.; Tentori, L.; Cappellozza, G.

This report is concerned with the evaluation of hematological parameters and of both relative (%) and absolute (mean pg/cell) quantities of the abnormal Hemoglobin (Hb) Hasharon in 53 heterozygous carriers and 7 double heterozygotes for Hb Hasharon and β-thalassemia from 43 apparently unrelated families living in the province of Rovigo (northern Italy). Biosynthetic studies are also reported. The data strongly suggest the presence of an α-thalassemia-2 determinant closely linked to the αHasharon-chain locus. Selective advantage of heterozygotes carrying such a-haplotype would explain the relatively high frequency of Hb Hasharon (0.23%) in northeastern Italy, a past-endemic malaria region. The interaction between Hb Hasharon and β-thalassemia results in preferential decrease of the abnormal Hb level. © 1980 S. Karger AG, Basel.

1980 - Post-translational control of human hemoglobin synthesis The role of the differential affinity between globin chains in the control of mutated globin gene expression [Articolo su rivista]
Mavilio, F.; Marinucci, M.; Guerriero, R.; Cappellozza, G.; Tentori, L.

The interactions between β-thalassemia and the humen hemoglobin (Hb) α-chain variants, Hb Hasharon, Hb O Indonesia and Hb J Paris, and between α-thalassemia and the β-chain variants, Hb S, Hb C and Hb G San José, which are characterized by preferential decrease of the abnormal Hb level in peripheral blood, have been studied. Both biosynthesis studies in reticulocytes and determination of the relative affinity of abnormal chains for normal complementary chains by in vivo recombination experiments, involving globin chains previously isolated in their native form, have been carried out in order to provide insights on the molecular events following the synthesis of the mutant chains under conditions of complementary chain deficiency. Furthermore, we have measured the relative affinity for complementary chain of βD Los Angeles- and αJ Rovigo-chains, the level of which does not decay in thalassemic carriers, and of αLegnano- and βOsu Christiansborg-chains, which have not yet been observed in association with thalassemias. Our experiments indicated that the differential affinity for β-chains is not always the major post-translational control mechanism which regulates the level of certain α-chain variants in β-thalassemic heterozygotes, and that preferential removal of abnormal chains by proteolytic enzymes is likely to play an important role in most cases. On the other hand, the low affinity of certain variant β-chains for α-chains may offer an explanation for the low level of certain β-chain variants in peripheral blood of non-thalassemic carriers, as well as to their decrease under conditions of relative α-chain deficiency (α-thalassemias). © 1980.

1979 - A new abnormal human hemoglobin: Hb prato (α231 (B12) Arg→Ser β2) [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Massa, A.; Gabbianelli, M.; Fontanarosa, P. P.; Camagna, A.; Ignesti, C.; Tentori, L.

An abnormal human hemoglobin was found in a hemolysate from a 5-year-old healthy child living in Prato (Tuscany, Italy). Structural studies demonstrated a previously unreported amino acid substitution, α31 (B12) Arg → Ser (this is an α 1β1 contact). The new variant has been named Hb Prato. It was unstable in isopropanol and heat-denaturation tests, but has normal functional properties, with respect to whole blood studies. Family studies indicated that the variant had been inherited from the mother, a 39-year-old woman of Sicilian extraction. Hb Prato occurs at 20 and 28% in hemolysates from the boy and woman, respectively. © 1979.

1979 - Genetic polymorphism of the α-globin haplotypes in a population from Calabria (Southern Italy). Studies with variants of the α-globin chain [Articolo su rivista]
Modiano, G.; Brancati, C.; Marinucci, M.; Massa, A.; Mavilio, F.; Tentori, L.

Four unrelated Calabrian families in which an α(J)-globin chain variant segregated have been studied. Structural studies identified the variant as Hb Norfolk (α2 57 (E6) Gly → Asp β2) in 1 family, and Hb J Oxford (α2 15 (A13) Gly → Asp β2) in 3 families. The abnormal hemoglobin has been quantitated both in absolute and relative terms in all the carriers in order to obtain information on the possible heterogeneity among the normal α(A)-haplotypes. The findings in 1 family strongly suggest that 2 quantitatively different α-haplotypes segregate. This provides evidence for the occurrence of a quantitative polymorphism of the α-haplotypes also in this population.

1979 - Haemoglobin Lepore Trait: Haematological and Structural Studies on the Italian Population [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Massa, A.; Gabbianelli, M.; Fontanarosa, P. P.; Samoggia, P.; Tentori, L.

Summary. Haematological data on 59 heterozygotes for haemoglobin (Hb) Lepore and 10 double heterozygotes for Hb Lepore and β thalassaemia from 36 Italian families are reported. The red cell indices are defined and compared with those of groups of non‐thalassaemic and β thalassaemic subjects of comparable number, age and sex distribution. The relative level of each haemoglobin fraction and the absolute production of single polypeptide chains are calculated in order to compare the expression of the non‐α chain genes in Hb Lepore trait and β thalassaemia. Structural studies demonstrate that the haemoglobin Lepore is of the Boston type (δ87β116) in all subjects, confirming that this type of fusion variant is probably the only one which occurs in Mediterranean populations. The distribution and incidence of the Lepore haemoglobinopathy are discussed. Copyright © 1979, Wiley Blackwell. All rights reserved

1979 - Hb j Baltimore (β 16 (a13) gly {right arrow, tailed} asp) in association with βthalassemia in a sicilian family [Articolo su rivista]
Musumeci, S.; Schiliro, G.; Fisher, A.; Musco, A.; Maritiucci, M.; Mavilio, F.; Fontanarosa, P. P.; Tentori, L.

1979 - Occurrence of haemoglobin norfolk (α257(E6) Gly→ASPβ2) at the level of 33% in an Italian family from calabria [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Samoggia, P.; Tentori, L.; Spadea, G.; Cocone, G.

An abnormal, fast-moving haemoglobin was observed in 5 healthy subjects of a family from Calabria (southern Italy). In all these carriers the abnormal haemoglobin, which structural studies identified as Hb Norfolk (α257(E6) Gly→Aspβ2) [4], occurs at a level averaging 33% of the total haemoglobin. Biosynthetic studies showed no evidence for unbalance of the globin chain synthetic ratio. In order to account for the observed percentages of Hb Norfolk, current concepts about the a-globin chain genetic system are reviewed, and different genie arrangements which would be in agreement with the experimental findings are discussed. © 1979 S. Karger AG, Basel.

1979 - Occurrence of hb j Paris in an italian family and recombination studies on the free abnormal αchain [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Tentori, L.; Bestetti, A.

1979 - Studies on a family with HB j calabria (α2 β2 64 (e8 GLY → ASP) [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Fontanarosa, P. P.; Tentori, L.; Brancati, C.

Hb J Calabria is a fast moving hemoglobin variant which was found in an Italian family by Vecchio et al. (1), and in a French family by Blouquit et al.who studied its functional properties (2). The original family described by Vecchio et al. in which both Hb J Calabria and βthal-assemia were present has been reexamined and is the subject of the present study. Hematological and clinical features of the carriers are described. The heterozygous carriers of Hb J Calabria showed only mild variable sub-clinical anemia and levels of the abnormal hemoglobin ranging from about 33 to 42% The Hb J Calabria/βthal-assemia double heterozygote showed a moderate chronic hemolytic anemia with alterations of the RBC indices and morphology in addition to splenomegaly. The relationship between structural abnormality, functional properties and clinical expression of Hb J Calabria is discussed. © 1979 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1979 - Synthesis of HB lepore Boston in peripheral blood [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Gabbianelli, M.; Tentori, L.; Isacchi, G.; Ialongo, P. L.; Papa, G.; Mandelli, F.

Two sisters, double heterozygotes for Hb Lepore and βthalassemia, of Italian origin (Naples), were extensively studied because of their particularly mild clinical and hematological picture, rather unusual in this type of syndrome. Family studies were carried out, and two Hb Lepore heterozygous carriers (the mother and a sister) and two βthalassemia heterozygotes (the father and a brother) were identified. In vitro biosynthetic studies carried out in both a Hb Lepore carrier and the Hb Lepore/βthalassemia double heterozygotes indicated an evident incorporation of radioactivity into the chromatographic peak which structural studies demonstrated to be Boston (= Washington) type of δbeta;chain (i.e., δ87 Gln β116 His). The amounts of synthesis were roughly of the same order of magnitude as the relative levels of Hb Lepore in the red cell hemolysates, indicating the presence, in the reticulocytes of these subjects, of stable δbeta; mRNA molecules. These findings provide the opportunity to reconsider the current views indicating the instability of mRNAs as the cause of the low synthetic activity of the δbeta; and βdelta; fusion genes. © 1979 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1978 - Haemoglobin lepore in Italy: Haematological and structural studies [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Massa, A.

This paper records the mean haematological and haemoglobin data and the structural characterization of 43 Hb Lepore heterozygotes from 27 apparently unrelated families detected either during screening programs or during haematological investigations of patients in the following parts of Italy: 1. Sicily (12 Hb Lepore heterozygotes from 10 apparently unrelated families); 2. Calabria (12 Hb Lepore heterozygotes from 4 apparently unrelated families); 3. Pyglia (2 related Hb Lepore heterozygotes); 4. Basilicata (3 related Hb Lepore heterozygotes); 5. Campania (7 apparently unrelated Hb Lepore heterozygotes); 6. Abruzzo (4 Hb Lepore heterozygotes from 2 apparently unrelated families); 7. Marche (2 related Hb Lepore heterozygotes); 8. Tuscany (1 Hb Lepore heterozygote).

1978 - Haemoglobin prato: A new amino acid substitution (α 31 (B12) ARG → SER) [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Massa, A.

1978 - Hb o Indonesia (α2 116(gh4) glu → lys β2): In association with βthalassemia [Abstract in Rivista]
Marinucci, M.; Mavilio, F.; Tentori, L.; Alberti, R.

1978 - Hemoglobin O Indonesia (alpha 2 116 (GH4) Glu leads to Lys beta 2) associated with beta-thalassemia in a family from Polesine (Italy) [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Tentori, L.; Alberti Mariuzzi, R. A.

1978 - Hemoglobin legnano (α2 141 (HC3) arg → leu β2): A new abnormal human hemoglobin with high oxygen affinity [Articolo su rivista]
Mavilio, F.; Marinucci, M.; Tentori, L.; Fontanarosa, P. P.; Rossi, U.; Biagiotti, S.

An abnormal, fast moving hemoglobin was noted in a 34-yr-old male patient living in Legnano (northern Italy) affected with renal failure and iron deficiency anemia, not related to the presence of the Hb variant. Structural studies have demonstrated a previously undescribed amino acid substitution, α 141 Arg → Leu. This new variant has been named Hb Legnano, and is characterized by an increased oxygen affinity and a low cooperativity, at least as far as preliminary functional studies carried out on whole blood have indicated. Family studies are reported: three other heterozygous carriers were observed among the relatives of the propositus, all showing a mild polycythemia which, however, does not seem to produce appreciable clinical consequences. © 1978 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1977 - Hemoglobin gavello - α2β2 47(CD6) asp → gly a new hemoglobin variant from polesine (Italy) [Articolo su rivista]
Marinucci, M.; Mavilio, F.; Tentori, L.; Alberti, R.

During a survey for abnormal hemoglobins in Polesine (a region north of the Po river, where betathalassemia is very frequent) a slow moving variant was noted in a 79-yr-old woman living in Gavello, a small town in the province of Rovigo. Structural studies demonstrated a previously undescribed amino acid substitution, 647 Asp à Gly. This new variant has been named Hb Gavello. © 1977 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.