Rapamycin-induced β-Globin Family Locus Remodeling Regulates the Gene Switching Expression / 中国生物化学与分子生物学报

β-Thalassemia caused by abnormal coding of the β-globin gene is the most common hemoglobinopathy in many Asian countries. The in-depth study of the molecular basis and epigenetic mechanism of globin gene expression is the key to explore a new treatment for thalassemia. In this study, FAIRE (formaldehyde-assisted isolation of regulatory elements), 3C (chromosome conformation capture) and ChIP (Chromatin Immunoprecipitation) were used to investigate the three-dimensional interaction network of β-globin family gene loci and the molecular mechanism of functional regulation of gene expression during rapamycin-induced chromatin remodeling in CD4+ T cells. The results showed that the opening degree of globin gene chromatin, the interaction frequency between the gene promoter region and the regulatory element LCR (Locus control regions), and the enrichment efficiency of CTCF (CCCTC-binding factor) in the gene promoter region changed differently during the change of rapamycin treatment concentration from low to high, which led to the same change trend of the gene expression pattern. At the 10 nmol/ L concentration, chromatin accessibility and gene expression decreased (P < 0. 05). At 20 nmol/ L and 50 nmol/ L concentrations, chromatin accessibility increased and gene expression was up-regulated (P < 0. 05). In this study, the molecular mechanism of gene expression regulation of the β-globin family was expounded through this dynamic change process. Our work provides a theoretical and clinical practice basis for clinical precision treatment.

Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy / 中国实验血液学杂志

OBJECTIVE@#To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin.@*METHODS@#The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34+ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test.@*RESULTS@#The human β-globin was expressed stably in the MEL cells, and CD34+ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34+ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant.@*CONCLUSION@#Stable β-globin expression through the optimization of HS4 from CD34+ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

Detection of β-globin Gene Mutations Among β-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System– Polymerase Chain Reaction

Background: β-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of β-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming. Methods: A simple polymerase chain reaction (PCR) approach involving a multiplex amplification refractory mutation system (MARMS) and one amplification refractory mutation system (ARMS), consisting of 20 β-globin gene mutations, were designed and employed to investigate β-thalassaemia patients and carriers. Results: Out of 169 carriers tested with the MARMS, Cd 41/42 (–TTCT), Cd 26 (A–G) HbE, IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 78.1%. Among the Malays, Cd 26 (A–G) HbE, Cd 41/42 (–TTCT), IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 81.4%, whereas Cd 41/42 (–TTCT) and IVS 2–654 (C–T) were most common among the Chinese (79.1%). Conclusion: We propose the use of this cheap, easy to interpret, and simple system for the molecular diagnostics of β-thalassaemia among Malaysians at the Institute for Medical Research (IMR)

Adeno-associated Virus Serotype 2 Mediated Transduction and Expression of The Human β-Globin Gene in Human Early Fetal Liver Hematopoietic Cells / 生物化学与生物物理进展

β-Thalassemia is an inheritance anaemia disease due to the defect in β- globin gene. Gene therapy is considered to be the only method which could cure this disease. Adeno-associated virus type 2 (AAV2) has benn gaining more attention as a vector in human gene therapy for its non pathogenic character and broad host range. Although, the efficacy of recombinant AAV2 (rAAV2) in transducing human hematopoietic stem cells has been investigated by researchers, the results were varied from different laboratory. The view was proposed recently that it may be resulted from helper virus in their packaging system. Respecting this, the packaging system without helper virus was used to produce rAAV2. Human early fetal liver hematopoietic cells not only possess many superior peculiarity compared to hematopoietic cells of bone marrow or cord blood, but also the inherent β-globin gene in the cells is not expressed. Studies on the AAV2 transduction of human fetal liver hematopoietic cells and mediated expression of β- globin gene in vivo were performed and the potential role of AAV2 in β-thalassemia gene therapy was analyzed. The rAAV2 containing a normal human β-globin gene (rAAV2-β-globin) without helper virus contamination were produced. The viral titer, purity and the ability of mediating expression of β-globin gene were detected in vitro. Then, human early fetal liver hematopoietic cells were isolated and were further transducted with the rAAV2-β-globin, followed by transplantation into sublethally irradiated BALB/C nude mice to analyze the β-globin gene expression. The results showed that the high titer and purity of rAAV2-β-globin had the ability of mediating β-globin gene expression in vitro. In 8 recipient BALB/C nude mice, the β-globin gene expression were detected in the 2 mice marrow by RT-PCR. The results suggested that rAAV2 could transduce human fetal liver hematopoietic cells and mediate the β-globin gene expression in BALB/C nude mice, meanwhile the expression level of the gene was still rather low. It is necessary to perform further research on AAV2 biology before applying in β-thalassemia gene therapy.