Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein ...Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.展开更多
Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cD-NA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained w...Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cD-NA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained which covered the entire cFIX coding region. Furthermore, GINaCcIX (driven by hCMV promoter) and GlNaMBcLX (driven by MCK enhancer and β-actin promoter) were constructed using the retroviral vector backbone of GINa. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/106 cell/24 h (GINaCcIX) and 211 ng/106 cell/24 h (GINaMBcIX) respectively. Those data offered a promising result for further animal study.展开更多
The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout)...The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.展开更多
基金supported by the State High Technology Development Program(863).
文摘Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.
基金Project Supported by the state High Technology Development Program
文摘Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cD-NA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained which covered the entire cFIX coding region. Furthermore, GINaCcIX (driven by hCMV promoter) and GlNaMBcLX (driven by MCK enhancer and β-actin promoter) were constructed using the retroviral vector backbone of GINa. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/106 cell/24 h (GINaCcIX) and 211 ng/106 cell/24 h (GINaMBcIX) respectively. Those data offered a promising result for further animal study.
基金Project supported by the State High Technology Development Program (863-Z20-02-01)Shanghai Postdoctoral Foundation and National Natural Science Foundation of China (Grant No. 39880019).
文摘The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.
