Recombinant adeno-associated virus (rAAV) vectors represent a promising approach to gene delivery for clinical use. Published data indicate that rAAV vector genomes persist in vivo as episomal chromatin in the skeletal muscle of nonhuman primates. In this study, we assessed the interconnection between the transcription factor cyclic AMP response element-binding protein (CREB) and recombinant AAV serotype 2 vector genomes after transduction in vitro and in vivo. rAAV-mediated myocyte transduction was potently blocked in the hearts of mice expressing CREB-S133A, which is a CREB-S133A dominant-negative mutant. Isoproterenol, a strong CREB activator, prominently increased rAAV transduction and the increase was abrogated by silencing the CREB gene with small interfering RNA. In addition, rAAV infection of muscle cells mildly but significantly induced CREB protein phosphorylation at serine-133, and was capable of stimulating CREB-dependent transcription from a reporter plasmid. Using chromatin immunoprecipitation and immunoblotting assays, both CREB and p300 were found to physically associate with two different rAAV genomes. Accordingly, CREB/p300 appears to have a role in rAAV transduction to establish active vector transcription in heart muscle cells.
Recombinant adeno-associated virus (rAAV)-based gene therapy represents a promising approach for the treatment of heart muscle diseases, but the molecular mechanisms that direct rAAV transduction remain unclear. Here we demonstrate that β-adrenergic receptor stimulation with isoproterenol (ISO) markedly increased cardiomyocyte transduction of rAAV in vitro and in vivo. Conversely, chronic β-adrenergic receptor downregulation significantly suppressed rAAV transduction. Pretreatment with calcium signaling cascade inhibitors including calcineurin inhibitory peptide (CNIP) strongly suppressed the positive effects of ISO on rAAV transduction. Additionally we document that ISO treatment led to a significant increase in double-stranded (ds) DNA synthesis of the rAAV genome and an increase in promoter activity. Moreover, stimulation with ISO did not affect rAAV transduction in calcineurin nullizygous mice. Collectively, we conclude that a calcium-dependent pathway regulates rAAV vector transduction at a number of stages that may include vector mobilization, conversion, and transcription activity. Modulating this pathway through β-adrenergic signaling enhances rAAV-mediated gene delivery to cardiomyocytes, and may be valuable when considering therapeutic approaches for heart muscle diseases.
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