Effects Of Liposomal Transfection Of Antisense Oligodeoxynucleotide On The Alpha-globin Of Major Beta-Thalassemia Erythroid Cells Cultured In Vitro

Beta-thalassemia major is a serious hereditary hemolytic anemia which does great harm to human being.It is a monogenic disease characterised by a reduced synthesis of beta-globin chains,which dues to the Doint mutation or deletion of beta-globin gene and its control region.It not only decreases the hemoglobin level,but breaks the balance of alpha/beta synthesis ratio.The major cellular pathogenetic mechanisms in beta thalassemia are based primarily on the deleterious effects produced by the accumulation of the excess alpha globin chain.These excess alpha-globin chains cannot form stable tetrameric structure and deposit in erythroid cells.They cause large ineffective erythropoiesis on their own,accelerate red cells destruction,and lead to hemolysis.Thus,inhibiting the endogenous alpha-globin genes and reducing chain unbalance may be a new strategy in the gene therapy of beta-thalassemia.Antisense technology can downregulate gene expression by targeting specific gene with antisense oligonucleotide(ASON).The previous reports have already indicated that ASON were potentially powerful and specific approach to downregulate gene expression.But there has been no report on targeting alpha-globin gene with ASON in beta-thalassemia.Based on the background above,we studied the effects of ASON on alpha-globin in beta-thalassemia major.The study consisted of three parts.In the first part,One-Step Erythroid Cells Liquid Culture System in Vitro was established.In the second part,the effective ASON and the optimal concentration targeting alpha-globin gene were screened after liposomal transfection into K562 cells.In the third part,the effects of liposomal transfection of ASON on alpha-globin in beta-thalassemia major erythroid cells cultured in vitro were observed.Our study focuses on the modification of alpha-globin chain production of beta-thalassemic erythroid cells on gene level, allmetioration of the unbalance of erythroid cells globin,and exploration of the new ideas of ASON technique for beta-thalassemia.1.Methods1.1 We established the 0ne-Step Erythroid Cells Liquid Culture System in Vitro.The cultured cells morphology,colony formation (includeing burst forming unit-erythroid,BFU-E and colony forming unit-erythroid,CFU-E),the cell ratio of displaying glycophorin A(GLA),the expression of fetal hemoglobin(HbF)and adult hemoglobin(HbA)were evaluated at different time points for learning the procedure of growth and differentiation of erythroid cells.1.2 On the basis of the PNA second structure,ASONs targeting alpha-globin gene were designed and synthesized relying on computed prediction.Further,the effective ASON and optimal concentration were screened after liposomal transfecting into K562 cells.The whole procedures were detected by Flow Cytometry for liposomal transfection efficiency,Real-Time PCR for alpha-globin gene expression,and Count Cell Kit-8 assay for the proliferation of K562 cells.1.3 The expression levels of alpha-,beta-and gamma-globin genes in normal and major beta-thalassemia were measured by Real-Time PCR.The effective ASON was transfected to the major beta-thalassemia erythroid cells cultured in vitro.The effects of liposomal transfection of ASON on the alpha-globin gene expression and the hemoglobin production were observed by Real-time PCR and HPLC respectively.In addition,the modify-cation of alpha-globin chain from ASON was objectively analyzed by electron microscope for the changes of precipitation of excess alpha-globin chains in erythroid cells cultured in vitro.2.Results2.1 Erythroid progenitor cells could gradually differentiate to mature erythrocyte in One-Step Liquid Culture in Vitro.The top of BFU-E and CFU-E were appeared on days 4th and 8th respectively. With the prolonged time in culture,high levels of GLA were detected,which tended to a steady increase to 10.7%,22.5%, 89.0%and 98.3%on days 0,4,8,12,respectively.A rise of HbF was seen from 0.5%on days 0 to 11%on days 12(P＜0.01).2.2.Liposomal could effectively transfect the FITC-labeled oligonucleotides into K562 cells.The transfection efficiencies were 62.3%,38.4%,25.8%at 24h,48h and 72h respectively.We designed 4 kinds of ASONs for targeting translated initiator and coding regions base on the computer prediction.The ASON consisting of 20 base pairs,which was against translated initiator,showed the effective inhibition efficiency.The effective ASON of inhibition of alpha-globin gene expression were dose dependent and time dependent.The gene expression levels of alpha-globin treated with 0.25μmol/L and 0.50μmol/L ASON group were 0.35±0.01 and 0.25±0.01(24h);0.24±0.01 and 0.21±0.01(48h),respectively(P＜0.01).The proliferation of K562 cell was obviously inhibited by oligonucleotide transfected with liposomal.The most proliferation inhibition was 83.32±0.75 in 0.50μmol/L ASON group(P＜0.01).Combined with the influence of ASON on the K562 cells proliferation,0.25μmol/L of ASON was determined as the optimal concentration.2.3 There were obvious unbalance between alpha-and beta-,gammaglobin gene expression in major beta-thalassemia.The ratio of alpha-and beta-,gamma-globin gene in major beta-thalassemia and normal control group were 2.22±0.25 and 1.11±0.07,respectively (P＜0.01).The ASON transfected by liposomal effective inhibited the expression of alpha-globin gene in major betathalassemic erythroid cells cultured in vitro.After treatment with ASON,the alpha-globin mRNA in ASON group and control group were 9.04±0.29 and 24.23±0.29,respectively(P＜0.01). Meanwhile,the disequilibrium of alpha-and beta-,gamma-globin gene was modified by ASON.The ratio of alpha-and beta-, gamma-globin gene in ASON group and control group were 0.79±0.02 and 2.26±0.06,respectivley(P＜0.01).The levels of HbA₂ and HbF increased in major beta-thalassemic erythroid cells after treatment with ASON.The HbF in major beta-thalassemic erythroid cells increased from 25.50±0.28 on 0 days to 66.05±3.32 on 12 days,but 35.25±3.04 was showed in control group on 12 days.The precipitates of alpha-globin chains in major beta-thalassemia erythroid cells were lessened after treatment with ASON,particularly in early erythroblast stage.There were no changes in those of control group. 3.ConclusionThe results showed that liposomal transfection of ASON effective inhibited the alpha-globin gene expression of major beta-thalassemic erythroid cells cultured in vitro,modified the ratio disqulibrium between alpha- and beta-,gamma-globin gene expression,reduced the precipitates of alpha-globin chains in erythroid cells.There was no such report before.Theoretically,ASON could inhibit the damage of relative excess of alpha chains and their degradation products to beta-thalassemic erythroid cells and prolong the life of abnormal erythroid cells.It may be very useful in the beta-thalassemia patients.It will provide a new way for the gene therapy in beta-thalassemia.