Evaluation Of β-thalassemia Therapeutic Effects Of Transgenic IPS Cells In Chimeric Mice

β-thalassemia is one of the most common genetic diseases, and is caused by aberrant versions of the β-globin gene. β-thalassemia mainly occurs in the Mediterranean, the Middle East and Southeast Asia. One-fifth of all β-thalassemia cases are β654 thalassemia in China, which is caused by C�'T substitution at β-globin nucleotide 654 of intron 2(βIVS–2–654) and results in aberrant RNA splicing with generation of trace amounts of nonfunctional polypeptide. The imbalance in the production of α- over β-globin activity leads to accumulation of free α-chain around erythrocyte membranes, intramedullary destruction of the erythroid precursors with ineffective erythropoiesis, and development of oxidative membrane damage associated with apoptosis in erythrocytes. The blood cells are destroyed during circulation, and the resulting myeloproliferative disorder is a general symptom in β-thalassemia patients.In China, since the end of mandatory premarital genetic testing, the birth rate of β-thalassemia babies has increased. Other than strengthening the use of pre-conception genetic testing to prevent this disease, there is no effective cure for β-thalassemia. Patient treatment mostly relies on life-long blood transfusion combined with iron chelation. Patients suffer long-term depletion of oxygen supply, and the visceral organs have pathological damage because of iron accumulation. β654 homozygous patients usually die at a very young age.At present, hematopoietic stem cell transfer could be an option for partially relieving symptoms, but limits in obtaining matched donor bone marrow is a hurdle, and bone marrow transplants can create two problems: immunological rejection and Graft Versus Host Disease(GVHD). The transduction of an extra copy of the normal human β-globin gene into allogenic hematopoietic stem cells was first demonstrated by May et al. In 2007, we concurrently used transgenic human β-globin combined with RNAi, or anti-sense RNA knockdown of α-globin for partial relief of anemia phenotypes in a mouse model. We also achieved effective treatment using in utero hematopoietic stem cell transplantation(IUHSCT). These approaches are prenatal, however, and would require diagnosis and treatment during gestation whereas most β654 thalassemia cases and associated anemia are only apparent after birth.Since the first induced pluripotent stem cells(iPSCs) of mice were generated in 2006, iPSCs have been considered good candidates for the replacement of malfunctional organs in future clinical applications. In 2007, one year after the introduction of induced pluripotent stem cells, Hanna et al. demonstrated that iPSCs could be used in treating sickle cell anemia in a mouse model. Two years later, we generated tetraploid complemented mice from iPSCs, confirming that iPSCs are totipotent. Recently, Chinese scientists successfully corrected human 41/42 β-thalassemia in iPSCs by homologous recombination. All these achievements shed light on strategies for treating some genetic diseases post-natally. In our research on iPSC-based treatments, we realized that there are three problems to be solved: safety, efficacy, and the dose threshold of treatment.To address these issues, we generated different iPS cell lines, including β654/LBG iPSCs(lentiviral vector LBG transduced β654 iPSCs) and HG-iPSCs(erythroidexpressing GFP iPSCs, derived from HS23-GFP transgenic mouse tail tip fibroblasts, these iPSCs had normal genotype). We then used chimeric mouse models to analyze how these iPSCs ameliorate the symptoms of β654 thalassemia. In our research, we generated four different chimeras: two kinds of β654/LBG iPSC chimeras, including Chimera-ICR mice(derived from ICR blastocysts) and Chimera-654 mice(derived from β654 blastocysts)and two kinds of HG-iPSCs chimeras, including GFP/β654 mice(derived from β654 blastocysts)and GFP/ICR mice(derived from ICR blastocysts). Herein we aim to evaluate the therapeutic effect of transgenic stem cells in treating β654 thalassemia in chimeric mice using iPS cells introduced into blastocytes.In all β654/LBG iPSCs chimeras, we proved that the transduced iPSCs could effectively differentiate into blood cells in vivo with effective expression of the human β-globin gene. The Chimera-ICR mice had almost no anemia phenotypes compared to their non-transduced siblings; even though chimerism exceeded 85%, diagnostic pathology examinations of blood, bone marrow and tissue sections showed no abnormalities. All Chimera-ICR mice had normal hematology parameters, including RBC, MCV, MCHC, and the size of spleens, which were the same as those of the wild type mice, implying that the β654/LBG iPSCs are safe and provide a stable gene compensation effect at the cell level. In Chimera-654 mice, the ratio of normally spliced β-globin RNA expression to abnormally spliced β-globin expression was different between different mice, and the higher the Chimera-654 chimerism, the higher the observed expression ratio. When the chimerism varied from 8% to 16% in the group Ch-654-1(Chimera-654 mice of low chimerism), splenomegaly and hematologic parameters still had typical thalassemia characteristics, but when the chimerism reached about 30% in the Ch-654-2 group(Chimera-654 mice of 31-37% chimerism), spleen mass and reticulocytes were reduced, and hemoglobin levels and RBC counts were increased accordingly. In our research, when the chimerism exceeded about 30%, the effective compensation for drawback gene’s function by β654/LBG iPSCs could be achieved.We also used normal HG-iPSCs without genetic defect to observe the amelioration of β-thalassemia. Here we chose chimeras with 10-30% chimerism. In these chimeras of low chimerism, HG-iPSCs also could differentiate into blood cells in vivo. The manifestations of β654-thalassemia, including iron accumulation in heart and spleen, splenomegaly, anemia, etc were alleviated to different extents. Compared to β654-thalassemia mice, hemoglobin levels(Hgb)(123.13±11.91, P<0.01) and Mean of Corpuscular Hemoglobin Concentration(MCHC)( 314.41±11.41, P<0.01) were increased in chimeras. This suggested that β654- thalassemia syndrome could be partially relieved in the chimeras of low chimerism.Our research confirmed both β654/LBG iPSCs and HG- iPSCs could effectively differentiate into blood cells, and can reverse the symptoms of β654- thalassemia. However the dose-dependent performance of these two iPSCs was different; HGiPSCs could achieve good curative effect with very low chimerism(10-30%), while at the same low chimerism, Chimera-654 mice still had pathological characteristics typical of β- thalassemia. β654/LBG iPSCs began displaying the compensation role only when the chimerism surpassed 30%. Chimera-654 mice had two different β-globin genes, normal splicing β-globin(derived from β654/LBG iPSCs) and abnormal splicing β-globin(derived from β654/LBG iPSCs and β654 blastocysts), so when the chimerism reached a threshold, the normal splicing β-globin could functionally compensate for the disease form of the gene, and β654- thalassemia syndrome could be partially relieved.In conclusion, we confirmed iPSCs could differentiate into blood cells effectively in vivo using chimera models, all the Chimera-ICR generated mice appeared to have no anemia phenotypes, while Chimera-654 mice showed increasing curative effect with rising chimerism levels, and all the chimeras had no tumors. This indicated β654/LBG iPSCs were safe, gene function compensation is effective in vivo in Chimera-654 mice, and transgenic β654 iPSCs could hold potential for treating β-thalassemia syndrome. We must now address the problem of dose-dependent performance. How to treat genetic diseases with less iPSCs is a very important problem which will impact the use of iPSCs in clinical applications.