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 /10 6 · 24 h and 1.6 ± 0.3 μg/10 6 · 24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×10 10 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
StatePublished - Dec 1 2003
Externally publishedYes

Fingerprint

hemophilia
Gene transfer
Hemophilia B
blood coagulation factors
Factor IX
Blood Coagulation Factors
gene transfer
Adenoviridae
Human engineering
Gene expression
gene expression
Gene Expression
Genes
mice
immune response
3T3 Cells
Bleeding Time
gene
Viral Genome
viral proteins

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)

Cite this

A study of gene transfer and expression of human clotting factor IX in Hemophilia B mice mediated by mini-adenoviral vector. / Gao, Xiaobo; Ye, Chenbo; Shi, Ding; Chen, Li; Qiu, Xinfang; Xue, Jinglun.

In: Science in China, Series C: Life Sciences, Vol. 46, No. 6, 01.12.2003, p. 631-640.

Research output: Contribution to journalArticle

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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 /10 6 · 24 h and 1.6 ± 0.3 μg/10 6 · 24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×10 10 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.",
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AU - Qiu, Xinfang

AU - Xue, Jinglun

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AB - 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 /10 6 · 24 h and 1.6 ± 0.3 μg/10 6 · 24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×10 10 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.

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