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Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B
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《医学前沿（英文）》 2015年 第9卷 第1期 页码 90-99 doi: 10.1007/s11684-015-0390-2
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.
关键词： factor IX hemophilia B liver-specific regulatory elements hydrodynamic gene transfer
Gene therapy for hemophilia B mice with scAAV8-LP1-hFIX
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《医学前沿（英文）》 2016年 第10卷 第2期 页码 212-218 doi: 10.1007/s11684-016-0438-y
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.
关键词： hemophilia B AAV8 hFIX gene therapy
《医学前沿（英文）》 2022年 第16卷 第4期 页码 584-595 doi: 10.1007/s11684-021-0844-7
关键词： hemophilia A adeno-associated virus (AAV) human/rat hybrid factor VIII gene therapy dual chain strategy
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Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B
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