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

Abstract

Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose–dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 × 1011 vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal 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/promoter alone was 120 to 200 ng/mL in mice injected with 1 × 1011vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the -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 previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol.

Time course of gene expression after plasmid DNA gene transfer to the liver

“…In our system, the MFG promoter was inactivated rapidly, suggesting an effect of the viral transduction process or cis regulatory elements. Similar differences in the effects of transcriptional regulatory elements upon longevity and level of expression between different delivery routes have been observed in skeletal muscle51. It is interesting to note that the chimeric intron increases expression levels driven by the EF1 promoter about two‐fold on Day 1, but results in lower expression levels on Day 7.…”

Heterogeneity of the immune response to adenovirus‐mediated factor VIII gene therapy in different inbred hemophilic mouse strains

“…Plasma FVIII levels were dependent on the dose of Ad delivered, and, as the dose of Ad‐CMV‐cFVIII increased in C57xBAL(F1)‐FVIIIKO from 6.0 × 10 10 to 4.8 × 10 11 , FVIII expression levels at 1 week increased from 0.1 U/ml to 24 U/ml (data not shown). Sustained long‐term transgene expression using a tissue‐restricted promoter has been reported previously 32, 37, 38.…”

Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD)

“…60 Incorporation of a sequence from the skeletal actin promoter containing at least a muscle-specific enhancer and an enhancer-like element further improved muscle-derived expression of coagulation factor (FIX) from a CMV enhancer/promoter-driven expression cassette. 61 A ITR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel ITR split gene vectors currently being considered for genes too large to be packaged. 62 In our lab, we have extensively tested two muscle-specific promoters, one for MCK and the other for a-skeletal actin (HSA), in both germline and somatic gene transfer.…”