Correction of Hemoglobin Levels in a Heterozygous Humanized Mouse Model of Thalassemia after Fetal Gene Therapy

Abstract 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 thalassemia (Cooley's anemia; CA) was used in which heterozygous animals are affected by anemia, splenomegaly and extra-medullary hematopoiesis. At E13.5 a “GLOBE” vector (HIV-2 based lentiviral vector that incorporates a mini hemoglobin beta gene, the beta-globin promoter and HS2/3 β-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 hematopoiesis was examined in the spleen and liver using flow cytometry (CD71+/Ter119+ cells) and histo-pathological analysis. Gene expression of human and mouse alpha and beta globins, as well as human gamma globin was assessed by quantitative polymerase chain reaction (qPCR). High performance liquid chromatography (HPLC) was used to quantify the presence of human beta globin in the peripheral blood of treated animals. Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the ethical committee on animal experimentation at University College London. Results Compared to non-injected heterozygous pups (control), IUGT increased hemoglobin 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 hematocrit [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 hematopoiesis 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 (figure A) and reduced expression of human gamma globin in blood and bone marrow of IUGT offspring. HPLC analysis confirmed these findings at protein level (figure B). The average vector copy number in the liver was 0.1. Conclusions 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 hemoglobin, confirming successful prenatal correction of the genetic defect. Figure A. Quantification of human beta globin mRNA using qPCR (*p<0.01) Figure A. Quantification of human beta globin mRNA using qPCR (*p<0.01) Figure B. HPLC analysis of peripheral blood hemolysates of control versus IUGT treated. Figure B. HPLC analysis of peripheral blood hemolysates of control versus IUGT treated. Disclosures No relevant conflicts of interest to declare.