Advancing Gene Therapy Vectors for Thalassaemia

Title: Advancing Gene Therapy Vectors for Thalassaemia

Budget: 144,000 Euros total

Funding body: Cyprus Research Promotion Foundation (ΥΓΕΙΑ/ΒΙΟΣ0609(ΒΕ)/20)

Project coordinator: Dr Marina Kleanthous

Partners: Carsten W. Lederer1, Coralea Stephanou1, Petros Patsali1, Michael Antoniou2, Soteroula Christou3, Natalia Michaelidou4, Roberto Gambari5

1. Molecular Thalassaemia Dept, Cyprus Institute of Neurology and Genetics, Cyprus
2. King’s College London School of Medicine, UK
3. Thalassaemia Centre, Makarios Hospital, Ministry of Health, Cyprus
4. Thalassaemia Association of Cyprus (Παγκύριος Αντιαναιμικός Σύνδεσμος), Cyprus
5. Department of Biochemistry and Molecular Biology, University of Ferrara, Italy



Thalassaemia is a potentially lethal single-gene disorder endemic to Cyprus, with curative treatment options only for a small proportion of patients and with a high cost and reduced quality of life for those undergoing chronic palliative treatment instead. As a single-gene disorder of the haematopoietic system, the disease is an ideal target for correction by gene therapy (GT) based on lentiviral vectors (LVs).[1] We therefore propose the following.

1) Based on the GLOBE LV[2] modified to hold a β-globin T87Q variation for reliable follow-up,[3] we will use novel β-globin-expressing LVs engineered to co-express a) short hairpin RNAs (shRNAs) against inhibitors of potentially therapeutic endogenous γ-globin expression and b) shRNAs and antisense snRNA for the knock-down and repair, respectively, of specific thalassaemic mRNA variants that compete with therapeutic lentivirally expressed β-globin mRNAs.[4]

2) As an essential resource for the project and other ongoing research of thalassaemia, in collaboration with the Cyprus Ministry of Health and in close adherence to national ethics guidelines on sample acquisition and patient privacy, we will establish a tissue bank for research purposes of thalassaemic and normal BM-derived mononuclear cells.

3) Drawing on the tissue collection under (2), we will use the in vitro-model of BM-derived CD34+ haematopoietic progenitor cells for the functional analysis of vector variants. In vitro differentiation of transduced cells in liquid and semi-solid media[5] will allow the assessment of LV-derived expression, of γ-globin induction and, for samples carrying specific β-thalassaemia mutations, of the amelioration of phenotypes resulting from specific β-globin mRNA defects.

4) Vectors designed to repair or knock down IVS1-110 will be tested in a pertaining humanised murine model[6] in collaboration with PA4 (Gambari Group, Ferrara). For vector versions without dedicated mouse model we will use the murine HBBth3 thalassaemia model[7] for transplantation of transduced HSCs, to allow a full in vivo evaluation of long-term safety and basic therapeutic efficacy (without benefit from human-specific shRNA expression). To gauge systemic γ-globin induction and off-target effects of shRNA-mediated knockdown, shRNA vectors effective for γ-globin induction under (1) will be adapted to match the respective murine orthologous target and will be tested in a mouse line encoding the entire human β-globin locus.[8]

The proposed project will give new insights into haemoglobin regulation and thalassaemia therapy, provide preclinical data for customised LVs, continue the transfer of international expertise in GT of thalassaemia to Cyprus and is expected to lead to two peer-review research publications. Moreover, the project is part of a long-term strategic effort to establish a GT network for thalassaemia and other disorders in Cyprus, thus ensuring modern treatments of Cypriot and other patients in the region and strengthening the role of Cyprus as a knowledge and treatment base for the thalassaemias.



1. Rivella S, Sadelain M: Therapeutic globin gene delivery using lentiviral vectors. Curr Opin Mol Ther 2002, 4(5):505-514.

2. Miccio A, Cesari R, Lotti F, Rossi C, Sanvito F, Ponzoni M, Routledge SJ, Chow CM, Antoniou MN, Ferrari G: In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia. Proc Natl Acad Sci U S A 2008, 105(30):10547-10552.

3. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F, Down J, Denaro M, Brady T, Westerman K et al: Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 2010, 467(7313):318-322.

4. Breda L, Casu C, Casula L, Kleinert DA, Bianchi N, Prus E, Cartegni L, Fibach E, Gardner LB, Giardina PJ et al: Following Beta-Globin Gene Transfer, the Production of Hemoglobin Depends Upon the Beta-Thalassemia Genotype. ASH Annual Meeting Abstracts 2009, 114(22):978.

5. Roselli EA, Mezzadra R, Frittoli MC, Maruggi G, Biral E, Mavilio F, Mastropietro F, Amato A, Tonon G, Refaldi C et al: Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients. EMBO Mol Med 2010, 2(8):315-328.

6. Vadolas J, Nefedov M, Wardan H, Mansooriderakshan S, Voullaire L, Jamsai D, Williamson R, Ioannou PA: Humanized beta-thalassemia mouse model containing the common IVSI-110 splicing mutation. J Biol Chem 2006, 281(11):7399-7405.

7. Yang B, Kirby S, Lewis J, Detloff PJ, Maeda N, Smithies O: A mouse model for beta 0-thalassemia. Proc Natl Acad Sci U S A 1995, 92(25):11608-11612.

8. Peterson KR, Clegg CH, Huxley C, Josephson BM, Haugen HS, Furukawa T, Stamatoyannopoulos G: Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human beta-globin locus display proper developmental control of human globin genes. Proc Natl Acad Sci U S A 1993, 90(16):7593-7597.

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