Optimized human factor IX expression cassettes for hepaticdirected gene therapy of hemophilia B  

作　　者：Ru Zhang Qiang Wang Lin Zhang Saijuan Chen 

机构地区：[1]State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruifin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

出　　处：《Frontiers of Medicine》2015年第1期90-99,共10页医学前沿（英文版）

摘　　要：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-LPI-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.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-LPI-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 

分 类 号：Q786[生物学—分子生物学] Q785