RESEARCH PROFILE

Background

Oxygen transport is one of, if not the most important physiological processes necessary to human life from the embryonic to the adult stage. Hemoglobin, the protein that carries oxygen to all our cells, is produced by the beta-globin gene and stored in mature red blood cells called erythrocytes. Beta-thalassemia major or Cooley's anemia is a genetic disorder that arises as a result of mutations in the beta-globin gene and is one of the most common inherited forms of chronic anemia, with greatest incidence in Southeast Asia, the Middle East and Africa. Some patients with the milder intermedia form of the disease (beta-thalassemia intermedia) may not need regular blood transfusions, but patients affected by beta-thalassemia major must have them to sustain life. As a consequence, patients become iron overloaded. Iron overload remains the biggest threat to the health of patients with thalassemia.

Several years ago people assumed that the iron overload exhibited in beta-thalassemia was only due to transfusions alone. Later, experts began to notice that even patients with beta-thalassemia intermedia, who did not undergo transfusions, had higher-than-average iron stores. In fact, this excess of iron is the cause of many serious complications and can even prove fatal. Some of the organs most severely affected are the liver, the heart and the endocrine glands, leading to liver fibrosis, cirrhosis and cancer, heart failure, growth impairment, diabetes and osteoporosis. Iron absorption from the gut is probably dictated by the degree of ineffective erythropoiesis. No definitive cure for the patient affected by beta-thalassemia is currently available.

First project:

The iron loading in thalassemia depends on the volume of blood transfused and the amount accumulated from gut absorption. Gut absorption is increased in thalassemia intermedia, where no or irregular transfusions are given. In thalassemia major, increased absorption is inversely proportional to the mean post-transfusional hemoglobin. We identified the major contributors of dangerous iron overload in beta-thalassemia. We discovered that proteins controlling iron intake such as hepcidin (Hamp1), a key signaler of body iron levels, and controller of ferroportin (Fpn1), an iron-transporting molecule active in the gut, are imbalanced in beta-thalassemia. In this paradigm, the body allows too much iron to enter the body through the intestine, boosting iron to unhealthy levels. Our present goal is to modulate hepcidin levels in the blood to control iron absorption under control in both the major and intermedia forms of the illness.

Second project:

Erythropoiesis is the process of making mature red blood cells (erythrocytes) from erythroid precursors cells. Any acquired or inherited genetic defect that reduces the production of red blood cells leads to anemia. In beta-thalassemia, anemia is characterized by ineffective erythropoiesis and is often lethal. Ineffective erythropoiesis is poorly understood but is thought to be cause by the augmented number of erythroid cell precursors that fail to generate normal erythrocytes. In addition, in beta-thalassemia the erythroid precursor cells leave the bones (their natural environment) and invade and damage other organs such as the spleen and liver. We aim to better understand ineffective erythropoiesis and to develop new therapies for the treatment of beta-thalassemia after characterization of genes that are dysregulated in the thalassemic erythroid cells. We believe that understanding how erythroid precursors proliferate and survive or die in normal and pathological conditions could be extremely important to better understand and treat thalassemia.

Third project:

Stem cell-based gene transfer offers a potential means to cure severe congenital diseases such as beta-thalassemia. We are working on ways of allowing healthy genes to be successfully transferred into hematopoietic stem cells, leading to a cure for thalassemia and other hematological diseases using lentivirus-based gene transfer. Using a gene delivery system (a lentiviral vector named TNS9) that carries the human beta-globin gene it is possible obtaining long-term correction of mice affected by beta-thalassemia intermedia. This vector also rescues mice affected by beta-thalassemia major. Presently, we are using human patient cells in vitro to generate a preclinical study that will assess if these vectors could be utilized to cure beta-thalassemia. In addition, we are generating and evaluating new lentiviral vectors using genomic elements that prevent silencing, position effects and insertional mutagenesis.

---

CHILDREN'S CANCER & BLOOD FOUNDATIONS LABORATORIES
JAFFE GENETICS CENTER

Members of the laboratory

Gene therapy of beta-thalassemia
Laura Breda, Ph.D.

Iron gene transfer, iron gene expression, iron metabolism and ineffective erythropoiesis
Sara Gardenghi, Ph.D.
Franca Maria Marongiu, Ph.D.
Ilaria Libani, Ph.D. candidate
Pedro Ramos, Ph.D. candidate

Laboratory Assistants
Ella Guy
Kevin Wang, Ph.D.

Manager of the Children's Cancer & Blood Foundation Laboratories
Scott Kerns

Past members:
Raffaella Schiro, M.D. (ineffective erythropoiesis)
Lisa Zampiero, Ph.D. candidate (beta-globin gene therapy)
Noel E. Mensah-Bonsu M.D. (ineffective erythropoiesis)

---

We are collaborating with biochemist Robert Grady, Ph.D. and Patricia J. Giardina, M.D., Head of the Hematology/Oncology Division and Chief of the beta-thalassemia Program at WMC and Coordinator of the Thalassemia Clinical Research Network, who have on-going basic and clinical research investigations regarding thalassemia.

Studies in iron metabolism and erythropoiesis in thalassemia and hemochromatosis are undergoing in collaboration with:

Amy Chadburn, M.D. and Yifang Liu, M.D. - WMC, Department of Pathology and Laboratory of Medicine
Stefan Worgall, M.D., Ph.D. - WMC, Department of Pediatrics, New York
Maria de Sousa, M.D. - Iron Genes and Immune System (IRIS Lab), IBMC - Instituto de Biologia Molecular e Celular, Oporto University, Oporto, Portugal
Gideon Rechavi, M.D., Ph.D. and Ninette Amariglio, Ph.D - Sheba Cancer Research Center (SCRC), Tel-Hashomer, Israel
Johannes Gerdes, Ph.D. and Thomas Scholzen Ph.D. ? Department of Immunology and Cell Biology, Research Center Borstel, Germany
Eliezer Rachmilewitz, M.D. - Hematology Institute, Wolfson Medical Center, Holon, Tel Aviv, Israel
Matteo Porotto, Ph.D. - WMC, Department of Pediatrics, New York
Tomas Ganz, M.D. and Ella Nemeth, Ph.D. - Departments of Medicine and Pathology, David Geffen School of Medicine, UCLA, Los Angeles
Nancy Andrews, M.D., Ph.D. and Cindy Roy, Ph.D., Department of Pediatrics, Harvard Medical School Children Hospital-Harvard, Boston
Nica Cappellini, M.D. ? Dept. of Internal Medicine, University of Milan, Milan, Italy

Bone abnormalities studies in beta-thalassemia are undergoing in collaboration with:

Maria Vogiatzy M.D. ? WMC, WMC, Department of Pediatric, New York
Adele Boskey, M.D. ? Mineralized Tissue Laboratory, Hospital for Special Surgery, New York

Gene transfer studies for the cure of beta-thalassemia are undergoing in collaboration with:

Michel Sadelain, M.D., Ph.D. and Luca Cartegni, Ph.D. at Memorial Sloan Kettering
Eitan Fibach, Ph.D. at the Hadassah University Hospital, Ein-Kerem in Jerusalem
Roberto Gambari, Ph.D. at the University of Ferrara in Italy.

Angiogenesis, stem cells and cancer:

David Lyden, M.D., Ph.D. - WMC, Department of Pediatrics, New York
Shahin Raffi, M.D., Ph.D. - WMC, Department of Medicine, New York
Paolo Mignatti, M.D. New York University, New York