A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eu...A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.展开更多
Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) vectors.Inhibited sec...Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) vectors.Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain.Previously,we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene,suggesting that the light chain,covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII.However,its level of secretion was affected by inefficient secretion the heavy chain.Here,we studied the effect of a mutant heavy chain with L303E/F309S substitutions,which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene.Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells.A spliced FVIII protein was seen in co-transfected cells by Western blot analysis.The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene,which was greater than the secretion of wild-type heavy chain.The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL,with an activity of (0.61±0.08) IU/mL,which was greater than that of wild-type FVIII co-transfected cells.Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections.This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector.These results encourage the transfer of this technology 展开更多
<strong>Background: </strong>ABO blood group distribution defers with racial and geographic variations. They are related to diseases like cardiovascular diseases, cerebral thromboembolism. ABO blood group ...<strong>Background: </strong>ABO blood group distribution defers with racial and geographic variations. They are related to diseases like cardiovascular diseases, cerebral thromboembolism. ABO blood group system may influence coagulation factor VIII which may increase the future risk of thrombosis. <strong>Aim:</strong> To assess the relation of ABO blood group with coagulation factor VIII in healthy adults.<strong> Material and Methods: </strong>A prospective type of analytical cross-sectional study was conducted in the Department of Physiology, Dhaka Medical College, Dhaka from July 2019 to June 2020. After obtaining ethical clearance, a total of 190 healthy adults were selected from different areas of Dhaka city based on inclusion and exclusion criteria, with ages ranging from 18 - 45 years. The subjects were interviewed and detailed history regarding personal, family, medical and drug were taken. Prior to sample collection, informed written consent was taken from the participants. Individuals of blood group A were selected as group A, blood group B as group B, blood group AB as group AB and blood group O as group O. Coagulation factor VIII was measured in the Department of Hematology and BMT Unit, Dhaka Medical College Hospital, Dhaka. Blood grouping was done in the Department of Physiology, Dhaka Medical College, Dhaka. <strong>Statistical Analysis:</strong> For statistical analysis, ONE way ANOVA followed by Bonferroni test were considered using SPSS 25.0 version. <strong>Results: </strong>In this study, blood group B was most common (33.2%). Coagulation factor VIII was significantly higher (p < 0.001) in blood group A (105.76% ± 11.82%), B (112.00% ± 15.02%), AB (109.80% ± 11.93%) than blood group O (82.00% ± 12.86%). No significant difference was observed among A, B and AB blood groups regarding coagulation factor VIII. <strong>Conclusions:</strong> It can be concluded that blood group A, B, AB individuals may have more chance of thrombosis due to significantly higher coagulation factor VIII than bloo展开更多
基金supported by the Natural Science Foundation of Shandong Province, China (Grant No. Y2005D14)the Science and Technology Program of Yantai (Grant No. 2008152)+1 种基金the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (Grant No. 20071108)the Discipline Construction Funds of Ludong University
文摘A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.
基金supported by the Natural Science Foundation of Shandong Province,China(ZR2010CM061)the Science and Technology Program of Yantai(2008152)the Scientific Research Foundation of Ludong University(LZ20083305)
文摘Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) vectors.Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain.Previously,we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene,suggesting that the light chain,covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII.However,its level of secretion was affected by inefficient secretion the heavy chain.Here,we studied the effect of a mutant heavy chain with L303E/F309S substitutions,which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene.Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells.A spliced FVIII protein was seen in co-transfected cells by Western blot analysis.The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene,which was greater than the secretion of wild-type heavy chain.The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL,with an activity of (0.61±0.08) IU/mL,which was greater than that of wild-type FVIII co-transfected cells.Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections.This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector.These results encourage the transfer of this technology
文摘<strong>Background: </strong>ABO blood group distribution defers with racial and geographic variations. They are related to diseases like cardiovascular diseases, cerebral thromboembolism. ABO blood group system may influence coagulation factor VIII which may increase the future risk of thrombosis. <strong>Aim:</strong> To assess the relation of ABO blood group with coagulation factor VIII in healthy adults.<strong> Material and Methods: </strong>A prospective type of analytical cross-sectional study was conducted in the Department of Physiology, Dhaka Medical College, Dhaka from July 2019 to June 2020. After obtaining ethical clearance, a total of 190 healthy adults were selected from different areas of Dhaka city based on inclusion and exclusion criteria, with ages ranging from 18 - 45 years. The subjects were interviewed and detailed history regarding personal, family, medical and drug were taken. Prior to sample collection, informed written consent was taken from the participants. Individuals of blood group A were selected as group A, blood group B as group B, blood group AB as group AB and blood group O as group O. Coagulation factor VIII was measured in the Department of Hematology and BMT Unit, Dhaka Medical College Hospital, Dhaka. Blood grouping was done in the Department of Physiology, Dhaka Medical College, Dhaka. <strong>Statistical Analysis:</strong> For statistical analysis, ONE way ANOVA followed by Bonferroni test were considered using SPSS 25.0 version. <strong>Results: </strong>In this study, blood group B was most common (33.2%). Coagulation factor VIII was significantly higher (p < 0.001) in blood group A (105.76% ± 11.82%), B (112.00% ± 15.02%), AB (109.80% ± 11.93%) than blood group O (82.00% ± 12.86%). No significant difference was observed among A, B and AB blood groups regarding coagulation factor VIII. <strong>Conclusions:</strong> It can be concluded that blood group A, B, AB individuals may have more chance of thrombosis due to significantly higher coagulation factor VIII than bloo
基金supported by the Natural Science Foundation of Shandong Province,China(No.Y2005D14)Science and Technology Program of Yantai municipality(No.2008152)+1 种基金the Scientific Research Foundation of Education Ministry for the Returned Overseas Chinese Scholarsthe Discipline Construction Funds of Ludong University
