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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (2) : 212-218     DOI: 10.1007/s11684-016-0438-y
Gene therapy for hemophilia B mice with scAAV8-LP1-hFIX
Wei Lu1,Qingzhang Zhou2,3,Hao Yang1,Hao Wang1,Yexing Gu1,Qi Shen1,Jinglun Xue1,Xiaoyan Dong2,*(),Jinzhong Chen1,*()
1. State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China
2. Beijing Five Plus Molecular Medicine Institute, Beijing 100176, China
3. College of Life Sciences, Jilin University, Changchun 130012, China
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Hemophilia B is a hemorrhagic disease caused by the deficiency of clotting factor IX (FIX). Gene therapy might be the ultimate strategy for the disease. However, two main problems that should be solved in gene therapy for hemophilia B are immunity and safety. Self-complementary adeno-associated virus serotype 8 (scAAV8), a non-human primate AAV featuring low immunogenicity and high transfection efficiency in liver cells, might be a potential vector for hemophilia B gene therapy. A strong liver-specific promoter-1 (LP1) was inserted and mutant human FIX Arg338Ala was introduced into plasmid scAAV8-LP1 to develop an optimized AAV8 vector that expresses human clotting factor FIX (hFIX). The efficiency of scAAV8-LP1-hFIX administered through normal systemic injection or hydrodynamic injection was compared. A high expression was achieved using hydrodynamic injection, and the peak hFIX expression levels in the 5×1011 and 1×1011 virus genome (vg) cohorts were 31.94% and 25.02% of normal level, respectively, at 60 days post-injection. From the perspective of long-term (200 days) expression, both injection methods presented promising results with the concentration value maintained above 4% of normal plasma. The results were further verified by enzyme-linked immunosorbent assay and activated partial thromboplastin time. Our study provides a potential gene therapy method for hemophilia B.

Keywords hemophilia B      AAV8      hFIX      gene therapy     
Corresponding Authors: Xiaoyan Dong,Jinzhong Chen   
Just Accepted Date: 16 March 2016   Online First Date: 06 April 2016    Issue Date: 27 May 2016
URL:     OR
Fig.1  Southern blot analysis of viral genomes of scAAV8-LP1-hFIX vectors. Genome of scAAV8-LP1-hFIX was characterized by Southern blot in lane1, and the 4900 nt ssDNA marker was in lane2.
Fig.2  Expression of hFIX after the injection of rscAAV8-LP1-FIX in hemophilia B mice (n = 4 per cohort). Plasma human FIX (hFIX) level (mean±SEM) measured by enzyme-linked immunosorbent assay (ELISA) at indicated time points (7, 14, 21, 30, 60, 90, 150, and 200 days post-injection) following the injection of rscAAV8-LP1-FIX with two injection methods (n = 4 in each cohort). (FH) 5 × 1011 vg by hydrodynamic injection; (FM) 1 × 1011 vg by hydrodynamic injection; (BH) 5 × 1011 vg by normal system injection; (BM) 1 × 1011 vg by normal system injection; (Control) PBS.
Fig.3  hFIX antibody level after the injection of rscAAV8-LP1-FIX in hemophilia B mice. Plasma hFIX antibody level measured by ELISA at indicated time points (7, 14, 21, 30, 60, 90, 150, and 200 days post-injection) following the hydrodynamic injection of rscAAV8-LP1-FIX. (FH) 5 × 1011 vg by hydrodynamic injection; (FM) 1 × 1011 vg by hydrodynamic injection; (BH) 5 × 1011 vg by normal system injection; (BM) 1 × 1011 vg by normal system injection; (Control) PBS.
Fig.4  Clotting time of all the experimental and wild-type cohorts at 30 days after injection. The tail was cut 1 cm, and filter paper was used to touch the wound on the tail every few seconds until no blood was shown on the filter paper. (FH) 5 × 1011 vg by hydrodynamic injection; (FM) 1 × 1011 vg by hydrodynamic injection; (BH) 5 × 1011 vg by normal system injection; (BM) 1 × 1011 vg by normal system injection; (control) PBS; (NA) hemophilia B mice; (WT) wild type.
Fig.5  Immunohistochemistry of hFIX-expressing hepatocytes in hemophilia B mice that received 5 × 1011 vg of rscAAV8-LP1-FIX vector and PBS 100 days post-injection. Magnification of all images is 100×. (A) Paraffin section from the liver of PBS treated mouse; (B) paraffin section from the liver of rscAAV8-LP1-FIX-treated mouse.
Fig.6  Histological examination of liver tissue that received 5 × 1011 vg of the rscAAV8-LP1-FIX vector and PBS by hydrodynamic injection. Animals following the hydrodynamic injection of 5 × 1011 vg and PBS were sacrificed on days 1 and 100. Magnification of all images is 100×. (A and C) Tissue from liver of rscAAV8-LP1-FIX-treated mouse; (A) 1 day; (C) 100 days. (B and D) Tissue from liver of PBS-treated mouse; (B) 1 day; (D) 100 days.
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