| Beta-hemoglobinopathies is caused by singleβ-globin genetic variation, of which the most frequent occurrence isβ-thalassemia and sickle cell disease. The patients with severe form maybe die without any treatment. For approaching the feasibility and potential value of gene RNA interference, the objective of this study is to design special RNAi target and to construct the lentiviral vector, to screen the effective target to specific bind and restrainβ-globin allele gene expression, and to observe the homing and repopulation of human infected CB CD34+ cells, and hope to conduce gene thrapy inβ-hemoglobinopathies by combination with other expression vector in follow-up experiment.Contents and Methods1. Based on the HBB mRNA sequence from GeneBank, we designed and selected target sequences for RNAi to HBB gene. After designed and synthesized, two single oligonucleotide DNA chains of each target were annealed and linked to enzyme lysised pGCL-GFP vector, then transferred to competence bacterial. The positive colony was amplified and extracted, and identified by PCR and sequencing.2. The relevantβ-globin gene fragment were copied and linked with pdsRED2-N1vector, then transferred to competence bacterial. The positive colony was amplified and extracted, and identified by PCR, sequencing and Blast analysis.3. HBB-RNAi-LV plasmid and HBB over-expression plasmid were cotransfected into 293T cells by lipofectamine 2000 with the low concentration group(0.5:1) and high concentration(1:1) experimental group. Cells were observed under fluorescence microscopy, and the Flag and HBB fusion protein were detected.4. Packaged lentivirus particles with HBB-RNAi-LV plasmid, pHelper 1.0 vector and pHelper 2.0 vector, the viral supernatant was then concentrated by Plus-20 ultrafiltration kit.5. CD34+ cells were isolated from human cord blood (CB) by MACS, cultured with cell factors and stroma-free SFM medium, and infected with MOI of 10 by LV particles. The ration of GFP positive cells was detected with FM and FCM. The HBB and HBG mRNA were detected by Real-Time PCR by 2, 4, or 5days after infection. The HBB protein level was determined by Western Blot.6. Human CB CD34+ cells prestimulated with high concentrated cell factors, and infected with LV virus, then injected into peritoneal cavity of SCID neonatal mice percataneously.7. Three weeks after transplantation, human HbF cells and adult red cells were deteced by FM and FCM in mouse blood. GFP positive cells were checked in both blood and bone marrow. GFP positive cells were also found in liver, speen, kidney and small intestine.Results1. Three HBB-RNAi-LV plasmids have been designed and constructed, named as PSC1, PSC2 and PSC3 respectively.2. The relevant HBB over-expression vector has been constructed, named as HBB- pdsRED2-N1-3Flag vectors.3. The RNAi effectiveness of PSC1 and PSC3 were confirmed by exogenous transfection experiment, and the effects were enhanced with the concentration of the RNAi plasmids increased, especially for PSC1.4. The high titer lentiviral virus of PSC1, PSC3 and PSCNC were obtained.5. After infection, the ratio of GFP positive cells was around 15~30% in CD34+cells. The HBB and HBG gene expressions were activated temporarily by lentivirus infection, and down-regulated relatively by PSC3 to which the degree of decrease is related to infection efficiency. In contrast, PSC1 up-regulated the HBB gene expression.6. After transplanted human infected CB CD34+ cells, percentage of human erythrocyte cells being 0.1-1.2% in peripheral blood, percentages of GFP positive cells being 4.4-8.8% vs 3.2-10.2%( PSC3 group vs PSCNC group) in peripheral nucleated cells, 1.3-6.2% vs 1.5-5.5%( PSC3 groups vs PSCNC groups) in bone marrow cells, there weren't statistical significance between groups. GFP positive cells were not founded in spleen, liver, kidney and small intestine tissues.Conclusions1. Establish a set of methods for separation, serum-free and stroma-free culture, and infection of human CB CD34+ cells. 2. Constructed three HBB-RNAi-LV vectors, confirmed the RNAi effectiveness of PSC1 and PSC3 by exogenous transfection experiment, packaged these two vectors and obtained high titer lentiviral virus.3. Obtained one efficient RNAi constrution for down-regulation HBB gene by infection human cord blood CD34+cells.4. After transplanted into nonmyelosuppressive SCID mouse, human CB CD34+cells with HBB shRNA may experience homing, repopulation and differentiation, in which lymphatic system were defective, and erythro-system being absence of competitive advantage.
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