The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) wa...The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by anintramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸexpression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸexpression curve. The QC-PCR method is easier and moreaccurate than traditional dot hybridization fordetermination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing inproduced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.展开更多
Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringers solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL ...Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringers solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression in-creased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene ex-pression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver dam-age, which was rapidly repaired within 3—10 d. These re-sults suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.30100102).
文摘The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which wasregulated by CMV promoter was constructed. Largequantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by anintramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸexpression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸexpression curve. The QC-PCR method is easier and moreaccurate than traditional dot hybridization fordetermination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing inproduced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.
文摘Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringers solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression in-creased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene ex-pression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver dam-age, which was rapidly repaired within 3—10 d. These re-sults suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.
