A study of gene transfer and expression of human clotting factor IX in Hemophilia B mice mediated by mini-adenoviral vector

Xiaobo Gao, Chenbo Ye, Ding Shi, Li Chen, Xinfang Qiu, Jinglun Xue

Research output: Contribution to journalArticle

Abstract

Vector Gti'IX 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 GTi'IX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTi'IX were obtained. At the same time, previous normal adenoviral vector pAdSPi'IX containing viral genome and hFIX mini-gene was constructed, and then previous adenovirus (pre-Ad) AdSPi'IX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTi'IX was less than 0.8%. 3T3 cells were transfected with AdGTi'IX and AdSPi'IX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1:4 ± 0.2 μg /106 · 24 h and 1.6 ± 0.3 μg/10 6 · 24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×1010 pfu AdGTi'IX or AdSPi'IX. 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 μL to 60 μL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTi'IX was obviously weaker than that triggered by AdSPi'IX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector system prolonged the expression time of hFIX and reduced immune response, thus offering a promising result for further pre-clinical study.

Original languageEnglish (US)
Pages (from-to)631-640
Number of pages10
JournalScience in China, Series C: Life Sciences
Volume46
Issue number6
DOIs
Publication statusPublished - Dec 1 2003
Externally publishedYes

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Keywords

  • Gene therapy
  • Hemophilia B
  • Hemophilia B mouse
  • Human clotting factor IX mini-gene
  • Mini-adenovirus

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Environmental Science(all)
  • Agricultural and Biological Sciences(all)

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