β-Thalassemia Gene Therapy Group

The Gene Therapy group has focused in the production of γ-globin lentiviral vectors for gene therapy of β-thalassemia. The β-thalassemias are inherited anemias that reduce or abolish production of the β-globin chain of hemoglobin. Caused by more than 200 mutations affecting the human β-globin gene, they are most prevalent in the Mediterranean region representing a serious health problem. The severity of β-thalassemia is directly linked to the degree of imbalance in the production of α and β like chains. The excess of α-globin chains that are not incorporated into adult hemoglobin (α2β2) precipitate in red blood cell precursors, impairing erythroid maturation and eventually causing apoptosis. Patients who suffer from β-thalassemia exhibit severe anemia that can be life threatening unless treated by blood transfusions. However the β-thalassemic phenotype is heterogeneous depending on the genotype as well as the degree of γ-globin chain expression. The beneficial effects of elevated fetal hemoglobin (α2γ2) in β-thalassemia have long been recognized because of amelioration of the clinical course of β-thalassemia syndromes. Patients who carry a β-thalassemia gene in deletional Hereditary Persistence of Fetal Hemoglobin (HPFH) are either asymptomatic or have milder clinical course. Hence, gene therapy of β-thalassemia based on γ-globin gene vectors has a considerable advantage. Our goal is to construct an ideal lentiviral vector that can produce therapeutic levels of γ-globin capable of ameliorating the effects of β-thalassemia and improve the patients' quality of life.

Designing novel therapeutic vectors for β-thalassemia gene therapy

Improving gene therapy protocols

Publications

Designing novel therapeutic vectors for β-thalassemia gene therapy

For β-thalassemia gene therapy to become an effective and realistic therapeutic approach, the following very important criteria need to be met:

1. The therapeutic vector should exhibit stability, high titers and erythroid-lineage specificity, through utilization of respective regulatory elements
2. The transgene must be expressed in therapeutic and sustained levels
3. The therapy itself should be safe and efficient in terms of viral transduction

Our group, generated a novel insulated SIN LV, designated as GGHI (Gamma-globin HPFH insulated), containing the Aγ-globin gene with the -117 HPFH point mutation and the HPFH-2 enhance This vector managed to produce efficient amounts of HbF, resulting in phenotypic correction in erythroid cultures of CD34⁺ HSCs isolated from the peripheral blood or bone marrow of thalassemic patients. In this specific vector design, the incorporation of the full length 1.2 kb cHS4 in the U3 region and the SIN configuration had no apparent effect on viral titer, as it reached 2x10⁸ TU/ml. GGHI is the first LCR-free LV that demonstrated efficient correction of β-thalassemia in vitro. GGHI is currently assessed also in vivo, in a murine model for β-thalassemia.

(Papanikolaou E, et al Human Gene Therapy 2012, Wilber A et al Blood. 2011)

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Improving gene therapy protocols

Based on the GGHI vector performance on thalassemic CD34⁺ cells in vitro, we aim to further improve the vector’s design, so as to induce higher transgene expression and ultimately β-thalassemia amelioration, both in vitro and in vivo using murine models of β-thalassemia. To this extent we are incorporating novel enhancer elements in GGHI, creating new γ-globin vectors that are currently under assessment for their performance in vitro, in CD34⁺ cells of normal and thalassemic individuals. These vectors are also destined for in vivo assessment in murine models of β-thalassemia.

The need for functional vectors’ generation, using fast, non-laborious and cost-effective strategies is imperative. We have developed an improved, quick protocol for preparing virus stocks destined for β-thalassemia gene therapy, which do not require any purification steps, yet yielding high titers, reaching more than 10⁹ TU/ml. Viruses produced with the revised ultrafiltration-based protocol are functional, since therapeutic vectors are contained in intact virions, and demonstrate reduced toxicity and high transduction efficiency upon transduction of both murine and human hematopoietic stem cells.

(Papanikolaou E, et al. Human Gene Therapy methods, submitted)

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Publications

1. Papanikolaou E, Georgomanoli M, Stamateris E, Panetsos F, Karagiorga M, Tsaftaridis P, Grafakos S and Anagnou, NP. The new self-inactivating lentiviral vector for thalassemia gene therapy combining two HPFH activating elements corrects human thalassemic hematopoietic stem cells. Human Gene Therapy 2012; 23(1): 15-31.
2. Wilber A, Hargrove PW, Kim YS, Riberdy JM, Sankaran VG, Papanikolaou E, Georgomanoli M, Anagnou NP, Orkin SH, Nienhuis AW, Persons DA. Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer. Blood. 2011; 117(10):2817-26.
3. Papanikolaou E, Kontostathi G, Drakopoulou E, Georgomanoli. M, Stamateris E, Vougas K, Vlahou A, Maloy A, Ware M. and Anagnou, NP.  Characterization and comparative performance of lentiviral vector preparations concentrated by one-step ultrafiltration. Human Gene Therapy Μethods, submitted