BACKGROUND: The development of a harmless and effi- cient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of ...BACKGROUND: The development of a harmless and effi- cient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of this study was to test a nonviral gene transferring vector's function of delivering DNA into liver cells to provide an important clue for gene transfer in liver gene therapy. METHODS: The complex of DNA and DNA delivering protein was injected into mice through their tail veins. Then the mice were killed and their liver tissue was sec- tioned. The gene transferring results were detected using a confocal laser scanning microscope. RESULTS: Fluorescence analysis indicated that both DNA- membrane penetrating peptide (MPP) complex and DNA- hepatocyte specific receptor binding domain ( HSRBD) - MPP complex could go into liver cells. The fluorescence value of liver cells in the DNA-HSRBD-MPP group was higher than that in the DNA-MPP group. CONCLUSIONS; MPP can successfully deliver DNA and protein into cells, and MPP with a HSRBD can specifically deliver DNA into liver cells. These have laid a foundation for further study on the nonviral liver cell gene delivering system.展开更多
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
Near-infrared （NIR） fluorescent metal nanodots may have significant advantages in biological detection and bioimaging. Herein, we introduce tunable near-infrared fluorescent gold nanodots （AuNDs） protected by bran...Near-infrared （NIR） fluorescent metal nanodots may have significant advantages in biological detection and bioimaging. Herein, we introduce tunable near-infrared fluorescent gold nanodots （AuNDs） protected by branched polyethylenimine （PEI） modified by surface segmental attachment of sulfhydryl groups （PEI-SH）, abbreviated as PEI-SH-AuNDs, for simultaneous gene delivery and cell imaging. The modified PEI endows the resultant PEI-SH-AuNDs with the following excellent advantages. Sulfhydryl groups of PEI-SH anchor to the surface of AuNDs, and such polycations with amine groups give PEI-SH-AuNDs remarkable stability. The cationic polymer PEI-SH with positive charges enables PEI-SH-AuNDs to perform gene delivery, and the gene transfection efficiency can reach 22.8%. Moreover, the fluorescence of PEI-SH-AuNDs is tunable from visible red light （wavelength 609 nm） to NIR light （wavelength 811 run） via an increase in the size of AuNDs. PEI-SH-AuNDs yielded gene transfection efficiency similar to that of commercial PEI, but showed much lower cytotoxicity and much greater red-shift fluorescence. With excellent photoluminescent properties, such multifunctional fluorescent PEI-SH-AuNDs hold promise in applications to bioimaging and as ideal fluorescent probes for tracking gene transfection behavior.展开更多
Protein trans-splicing based dual-vector factor VIII （FVIII） gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulati...Protein trans-splicing based dual-vector factor VIII （FVIII） gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide （442± 151 ng mL i vs. 305±103 ng mL-1） and coagulation activi- ty （1.46±0.37 IU mL i vs. 0.85±0.23 IU mL-1） compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector ex- pressing FVIII （1.79_＋0.59 IU mL-l）. These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.展开更多
Dr. Craig Mello, the Nobel Prize Winner of 2006 in Physiology or Medicine, delivered a speech entitled Return to The RNA World： Rethinking Gene Expression, Evolution, and Medicine at Tsinghua on March 26, 2007.
Hemophilia A is caused by a genetic mutation in coagulation factor VIII （FVIII） gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vect...Hemophilia A is caused by a genetic mutation in coagulation factor VIII （FVIII） gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII （BDD-FVIII） leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately （239＋_56） ng mL-1 above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately （1.09＋_0.25） IU mL-1 above en- dogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.展开更多
基金This study was supported by grants from the National Natural Science Foun-dation of China( No:30472251 )and the Shanxi Youth Science Fund ( No.020011028).
文摘BACKGROUND: The development of a harmless and effi- cient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of this study was to test a nonviral gene transferring vector's function of delivering DNA into liver cells to provide an important clue for gene transfer in liver gene therapy. METHODS: The complex of DNA and DNA delivering protein was injected into mice through their tail veins. Then the mice were killed and their liver tissue was sec- tioned. The gene transferring results were detected using a confocal laser scanning microscope. RESULTS: Fluorescence analysis indicated that both DNA- membrane penetrating peptide (MPP) complex and DNA- hepatocyte specific receptor binding domain ( HSRBD) - MPP complex could go into liver cells. The fluorescence value of liver cells in the DNA-HSRBD-MPP group was higher than that in the DNA-MPP group. CONCLUSIONS; MPP can successfully deliver DNA and protein into cells, and MPP with a HSRBD can specifically deliver DNA into liver cells. These have laid a foundation for further study on the nonviral liver cell gene delivering system.
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This work was supported by the National Natural Science Foundation of China （Nos. 51503085, 51373061 and 21304090）, Science Foundation of China University of Petroleum, Beijing （No. 2462017YJRC027）, open project of state key laboratory of supramolecular structure and materials （No. sklssm201724） and Graduate Innovation Fund of Jilin University （Project 2016112）.
文摘Near-infrared （NIR） fluorescent metal nanodots may have significant advantages in biological detection and bioimaging. Herein, we introduce tunable near-infrared fluorescent gold nanodots （AuNDs） protected by branched polyethylenimine （PEI） modified by surface segmental attachment of sulfhydryl groups （PEI-SH）, abbreviated as PEI-SH-AuNDs, for simultaneous gene delivery and cell imaging. The modified PEI endows the resultant PEI-SH-AuNDs with the following excellent advantages. Sulfhydryl groups of PEI-SH anchor to the surface of AuNDs, and such polycations with amine groups give PEI-SH-AuNDs remarkable stability. The cationic polymer PEI-SH with positive charges enables PEI-SH-AuNDs to perform gene delivery, and the gene transfection efficiency can reach 22.8%. Moreover, the fluorescence of PEI-SH-AuNDs is tunable from visible red light （wavelength 609 nm） to NIR light （wavelength 811 run） via an increase in the size of AuNDs. PEI-SH-AuNDs yielded gene transfection efficiency similar to that of commercial PEI, but showed much lower cytotoxicity and much greater red-shift fluorescence. With excellent photoluminescent properties, such multifunctional fluorescent PEI-SH-AuNDs hold promise in applications to bioimaging and as ideal fluorescent probes for tracking gene transfection behavior.
基金supported by the Natural Science Foundation of Shandong Province (ZR2010CM061)the Scientific Research Foundation from Ministry of Education for Returned Overseas Chinese Scholars (20071108)
文摘Protein trans-splicing based dual-vector factor VIII （FVIII） gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide （442± 151 ng mL i vs. 305±103 ng mL-1） and coagulation activi- ty （1.46±0.37 IU mL i vs. 0.85±0.23 IU mL-1） compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector ex- pressing FVIII （1.79_＋0.59 IU mL-l）. These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.
文摘Dr. Craig Mello, the Nobel Prize Winner of 2006 in Physiology or Medicine, delivered a speech entitled Return to The RNA World： Rethinking Gene Expression, Evolution, and Medicine at Tsinghua on March 26, 2007.
基金supported by the Natural Science Foundation of Shandong Province,China(Grant No.ZR2010CM061)the Scientific Research Foundation from Ministry of Education for Returned Overseas Chinese Scholars(Grant No.20071108)
文摘Hemophilia A is caused by a genetic mutation in coagulation factor VIII （FVIII） gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII （BDD-FVIII） leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately （239＋_56） ng mL-1 above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately （1.09＋_0.25） IU mL-1 above en- dogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.
