Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter

Hemophilia B is caused by the absence of functional coagulation factor IX ( F.IX) and represents an important model for treatment of genetic diseases b y gene therapy. Recent studies have shown that intramuscular injection of a n adeno-associated Viral (AAV) vector into mice and hemophilia B dogs resul ts in vector dose-dependent, long-term expression of biologically active F. IX at therapeutic levels. In this study, we demonstrate that levels of expr ession of approximately 300 ng/mL (6% of normal human F.IX levels) can be r eached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites ) than previously described. This was accomplished through the use of an im proved expression cassette that uses the cytomegalovirus (CMV) immediate ea rly enhancer/promoter in combination with a 1.2-kilobase portion of human s keletal actin promoter. These results correlated with enhanced levels of F. IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes , Systemic F.IX expression from constructs containing the CMV enhancer/prom oter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector gen omes, Muscle-specific promoters performed poorly for F.IX trans-gene expres sion in vitro and in vivo. However, the incorporation of a sequence from th e or-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previousl y published results. These findings will allow the design of a clinical pro tocol for therapeutic levels of F.IX expression with lower vector doses, th us enhancing efficacy and safety of the protocol. (Blood, 2000;95:2536-2542 ) (C) 2000 by The American Society of Hematology.