Presented at the Neonatal Society 2015 Spring Meeting.
Shangaris P1,2, Loukogeorgakis SP1,3, Subramaniam S1, Blundell M7, Bakhamis N2, Liu S4, Eaton S1, Ramachandra D1, Maghsoudlou P1, Urbani L1, Waddington S2, Archer J5, Antoniou M6, Thrasher AJ7, Ryan T5, De Coppi P1, David A2
1 Surgery Unit, Institute of Child Health, University College London, UK
2 Institute for Women’s Health, Maternal & Fetal Medicine, University College London, UK
3 Center for Fetal Research, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA
4 University of Alabama at Birmingham, Birmingham, AL, USA
5 Central Diagnostic Services, Queen’s Vet School Hospital, University of Cambridge, UK
6 Department of Medical and Molecular Genetics, King’s College London, UK
7 Molecular Immunology Unit, Institute of Child Health, UK
Background: Beta thalassaemia is a genetic blood disease that causes life-threatening anemia. Hematopoietic stem cell (HSC) transplantation successfully cures the disease but in only 30% of patients. We hypothesized that in utero gene therapy (IUGT) to the fetal HSC compartment with the corrected beta globin gene might cure the disease before birth.
Methods: A humanized mouse model of thalassaemia (Cooley’s anemia;CA) was used in which heterozygous animals are affected by anaemia, splenomegaly and extra-medullary haematopoiesis. At E13.5 a “GLOBE” vector (HIV-2 based lentiviral vector that incorporates a mini haemoglobin beta gene, the beta-globin promoter and HS2/3β-LCR element) was injected into the liver of each fetus (n=12). At 12 weeks of age, recipient blood, liver, spleen and bone marrow were collected for complete blood count, blood film, as well as RNA and DNA isolation. Extra-medullary haematopoiesis was examined in the spleen and liver using flow cytometry (CD71+/Ter119+cells) and histo-pathological analysis.
Results: Compared to non-injected heterozygous pups (control), IUGT increased haemoglobin levels [11.3±0.4g/dl (n=6) vs. 7.6±0.6g/dl (n=8); p<0.01], red blood cell count [9.3±0.3*1012/L vs. 6.2±0.5*1012/L; p<0.01), and haematocrit [41.2±2.2% vs. 27.2±2.0; p<0.01]. Moreover, treated CA animals had reduced spleen weight [130±5mg vs.310±21mg; p<0.01], as well as reduced extra-medullary haematopoiesis in the liver [0.7±0.1% (n=4) vs. 6.0±0.9% (n=5); p<0.01] and spleen [6.6±1.8 (n=4) vs. 23.1±1.4 (n=3); p<0.05]. qPCR analysis demonstrated increased gene expression of human beta globin and reduced expression of human gamma globin in blood and bone marrow of IUGT offspring. HPLC analysis confirmed these findings at protein level. The average vector copy number in the liver was 0.1.
Conclusion: IUGT resulted in phenotypic normalization in a heterozygous humanized mouse model of CA. Increased levels of beta globin and associated down-regulation of gamma globin is consistent with a switch from fetal to adult human haemoglobin, confirming successful prenatal correction of the genetic defect.
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