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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.    2015, Vol. 9 Issue (1) : 90-99     DOI: 10.1007/s11684-015-0390-2
Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B
Ru Zhang,Qiang Wang,Lin Zhang,Saijuan Chen()
State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Gene therapy provides a potential cure for hemophilia B, and significant progress has been achieved in liver-directed gene transfer mediated by adeno-associated viral vectors. Recent clinical trials involving the use of a self-complementary adeno-associated virus serotype 8-human codon-optimized factor IX (AAV8-hFIXco) vector demonstrated encouraging efficacy with hFIX expression stabilized at 1% to 6% of normal level in patients, but safety concerns related to high vector doses are still present. Thus, further improvement of AAV vectors and hFIX expression cassette may positively contribute to the ultimate success of hemophilia B gene therapy. In this study, to obtain a higher expression level of hFIX that potentiates the coagulant capacity of recipients, human FIX expression vector was optimized by upgrading the codon adaption index and adjusting the GC content, inserting a Kozak sequence (GCCACC), and introducing a gain-of-function mutation, R338L (FIX Padua). The efficiency of the published and the presently constructed cassettes was compared through in vivo screening. In addition, the regulatory elements that control the FIX gene expression in these cassettes were screened for liver-specific effectiveness. Among all the constructed cassettes, scAAV-Pre-hFIXco-SIH-R338L, which was the construct under the control of the prothrombin enhancer and prealbumin promoter, resulted in the highest level of coagulant activity, and the expression levels of two constructed cassettes (scAAV-Chi-hFIXco-SIH-R338L and scAAV-Pre-hFIXco-SIH-R338L) were also higher than that of the published cassette (scAAV-LP1-hFIXco-SJ). In summary, our strategies led to a substantial increase in hFIX expression at the protein level or a remarkably elevated coagulant activity. Thus, these reconstructs of hFIX with AAV vector may potentially contribute to the creation of an efficacious gene therapy of hemophilia B.

Keywords factor IX      hemophilia B      liver-specific regulatory elements      hydrodynamic gene transfer     
Corresponding Authors: Saijuan Chen   
Just Accepted Date: 16 January 2015   Online First Date: 10 February 2015    Issue Date: 02 March 2015
URL:     OR
Fig.1  Construction of hFIX expression cassettes. (A) Diagram of human FIX expression cassette: a liver-specific FIX expression cassette comprises FIX cDNA and liver-specific regulatory elements, including enhancer, promoter, intron, and polyA flanked by the AAV internal terminal repeats. The AAV internal terminal repeats were necessary for packaging the virion. (B) Each cassette had intact 5′ and 3′ ITRs. scAAV-LP1-hFIXco-SJ contained the LP1 promoter, which comprised core liver-specific elements from HCR and hAAT, modified SV40 intron, hFIXco-SJ, and late SV40 polyA [5]. scAAV-LP1-hFIXco-SJ-R338L, scAAV-LP1-hFIXco-SIH, scAAV-LP1-hFIXco-SIH-R338L, and scAAV-LP1-hFIXwt contained the same control elements as scAAV-LP1-hFIXco-SJ, and those cassettes had different hFIX as shown in the Figure above. scAAV-Alb-hFIXco-SIH-R338L, scAAV-Pre-hFIXco-SIH-R338L, and scAAV-Chi-hFIXco-SIH-R338L contained the same intron (Chimeric intron), polyA (Synthetic polyA), and hFIXco-SIH, which were driven by albumin enhancer (221 bp) and albumin promoter (292 bp), prothrombin/prealbumin enhancer (180 bp) and prealbumin promoter (216 bp), chimeric albumin enhancer (228 bp) and prealbumin/albumin promoter (215 bp), respectively.
Plasmid name Enhancer Promoter Intron hFIX PolyA
LP1-hFIXwt ApoE-HCR hAAT Mod-SV40 hFIXwt Late SV40
LP1-hFIXco-SJ ApoE-HCR hAAT Mod-SV40 hFIXco-SJ Late SV40
LP1-hFIXco-SIH ApoE-HCR hAAT Mod-SV40 hFIXco-SIH Late SV40
LP1-hFIXco-SJ-R338L ApoE-HCR hAAT Mod-SV40 hFIXco-SJ-R338L Late SV40
LP1-hFIXco-SIH-R338L ApoE-HCR hAAT Mod-SV40 hFIXco-SIH-R338L Late SV40
Alb-hFIXco-SIH-R338L Alb Alb Chimeric hFIXco-SIH-R338L Synthetic
Pre-hFIXco-SIH-R338L Pro/Prea Prealbumin Chimeric hFIXco-SIH-R338L Synthetic
Chi-hFIXco-SIH-R338L Alb/Albb Pre/Albc Chimeric hFIXco-SIH-R338L Synthetic
Tab.1  Structures of different FIX expression cassettes applied in this study
Fig.2  Codon optimization of FIX: adjustment of the CAI, GC content and the frequency of codon usage for improving FIX expression. (A) A CAI of 1.0 was considered as perfect, whereas a CAI of>0.8 was good, for high-level gene expression in the desired organism [30]. (B) The ideal range of GC content was between 30% and 70%. Peaks of GC content percentage in a 60 bp window were removed. (C) Frequency of optimal codons was the percentage distribution of codons in computed codon quality groups. The value of 100 was set for the codon with the highest usage frequency for a given amino acid in the desired expression organism.
Fig.3  Effects of codon optimization on hFIX expression under the control of the same elements. ELISA was used to detect plasma hFIX antigen at 36 h after tail-vein hydrodynamic administration of saline (CON, n = 3), scAAV-LP1-hFIX (FIX-WT, n = 5), scAAV-LP1-hFIXco-SIH (FIX-SIH, n = 8), and scAAV-LP1-hFIXco-SJ (FIX-SJ, n = 8). (A+ B) hFIX antigen levels in the plasma of individual mice are indicated in (A) and presented as mean±SD in (B). Saline-injected mice produced nearly undetectable hFIX antigen (0.2% of normal). FIX-WT, FIX-SIH, and FIX-SJ had a mean hFIX antigen levels of 47%±7% (2.35±0.14 μg/ml), 162%±15% (8.1±0.3 μg/ml), and 157%±19% (7.85±0.95 μg/ml) of normal, respectively.
Fig.4  Comparison of hFIX coagulant activity and expression level mediated by transgene cassettes in HB mice after hydrodynamic injection. FIX-SJ: scAAV-LP1-hFIXco-SJ; FIX-SJ-M: scAAV-LP1-hFIXco-SJ-R338L; FIX-SIH: scAAV-LP1-hFIXco-SIH; and FIX-SIH-M: scAAV-LP1-hFIXco-SIH-R338L. (A+ B) Chromogenic assay to determine hFIX activity in individual mice in (A) is presented as mean±SD in (B). The activity is calculated as a percentage of normal hFIX activity in the pooled plasma. The average hFIX activities mediated by FIX-SJ, FIX-SJ-M, FIX-SIH, and FIX-SIH-M were about 60%±24%, 406%±32%, 66%±16%, and 479%±37%, respectively. (C+ D) ELISA results of hFIX expression in individual mice in (C) are presented as mean±SD in (D). The expression of codon-optimized hFIX with or without R338L mutation showed no significant difference.
Fig.5  Comparison of liver-specific regulatory elements driving hFIX expressionin vivo after hydrodynamic delivery. (A+ B) Chromogenic assay to determine hFIX coagulant activity, which was calculated as percentage of normal hFIX activity in pooled human plasma. The activities in individual mice are indicated in (A) and presented as mean±SD in (B). LP1-SJ: LP1-hFIXco-SJ (n = 4); LP1-SIH-M: LP1-hFIXco-SIH-M (n = 5); Alb-SIH-M: Alb-hFIXco-SIH-M (n = 5); Chi-SIH-M: Chi-hFIXco-SIH-M (n = 4); and Pre-SIH-M: Pre-hFIXco-SIH-M (n = 5).
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[1] Supplementary Material Download
[1] Wei Lu,Qingzhang Zhou,Hao Yang,Hao Wang,Yexing Gu,Qi Shen,Jinglun Xue,Xiaoyan Dong,Jinzhong Chen. Gene therapy for hemophilia B mice with scAAV8-LP1-hFIX[J]. Front. Med., 2016, 10(2): 212-218.
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