StableIn VivoTransgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10

Abstract

Our long-term goal is to prevent or reverse atherosclerosis by delivering gene therapy from stably transduced endothelial cells (EC). We previously reported that EC-directed gene therapy with a helper-dependent adenovirus (HDAd) expressing apolipoprotein A-I (apo A-I) retarded development of atherosclerosis in rabbit carotid arteries over a 1-month interval. However, a 70% decline in apo A-I expression during this time raised concerns about long-term efficacy of this approach. Here we report use of several approaches aimed either at preventing this decline or at increasing apo A-I expression from HDAd at all time points: codon optimization, deletion of 3' untranslated sequences, substitution of a synthetic mammalian-based promoter (4XETE) for the cytomegalovirus (CMV) promoter, and co-transduction with an HDAd expressing interleukin-10. We tested these approaches using plasmid transfection of cultured EC and in vivo transduction of rabbit carotid artery EC. Codon optimization did not increase apo A-I expression. Deletion of 3' untranslated sequences extinguished apo A-I expression. Both substitution of 4XETE for the CMV promoter and expression of interleukin-10 stabilized apo A-I expression in vivo, although at the cost of lower early (3-day) expression levels. Surprisingly, both interventions stabilized apo A-I expression without altering the rate at which HDAd genomes were lost. These data establish that transgene expression from HDAd in EC is inherently stable in vivo and suggest that the early decline of CMV promoter-driven expression from HDAd-transduced EC is due neither to active downregulation of transcription nor to loss of HDAd genomes. Instead, apparent loss of expression from the CMV promoter appears to be a consequence of early (3-day) upregulation of CMV promoter activity via inflammatory pathways. Our results yield new paradigms to explain the early loss of genomes and transgene expression after in vivo gene transfer. These new paradigms will redirect strategies for achieving high-level, stable expression of transgenes in EC.

A Rabbit Model for Testing Helper-Dependent Adenovirus-Mediated Gene Therapy for Vein Graft Atherosclerosis

“…We believe that the decline in cytokine expression between 4 and 12 weeks is likely due to acute cytokine upregulation in 4-week grafts as a consequence of the recent surgery and vector infusion. 54 Because we are developing a therapy for vein graft atherosclerosis, rather than for surgery- and vector-related inflammation, 12 weeks again seems to be a better time point for experimental vein graft harvests.…”

“…HDAd genomes in the vein grafts declined substantially between 4 and 20 weeks after transduction, although they remained at a relatively high absolute level (∼4 vector genomes per endothelial cell). Genome loss during this interval may reflect disappearance of transcriptionally inactive genomes (as occurs between 3 and 28 days after transduction), 54 and loss of transcriptionally inactive genomes would not affect vector-derived transgene expression. Nevertheless, additional work is needed to determine whether HDAd can mediate long-lasting (i.e., years) transgene expression in atherosclerotic vein grafts.…”

“…We also found that HDAd has pro-inflammatory effects in vein grafts of chow-fed rabbits, even when it is infused at the relatively low dose of 2 × 10 11 particles/mL (a dose that does not significantly increase inflammation in arteries of fat-fed rabbits). 29 Whether these inflammatory effects will preclude use of HDAd for vein graft gene therapy and whether the pro-inflammatory effects might be decreased or eliminated either by expression of an anti-inflammatory cytokine such as IL-10 54 or by expression of a therapeutic transgene that has anti-inflammatory activity (e.g., apo A-I) 33 , 65 will require further testing. We look forward to performing future studies that answer these questions and further clarify the potential of HDAd to deliver durable and effective gene therapy for vein graft atherosclerosis.…”

Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

“…To begin development of tissue protein extraction techniques for enrichment of ECM proteins, aortic segments were obtained postmortem from a chow-fed rabbit that was euthanized in the course of unrelated experiments. 31 All other animal experiments were performed with mice. All mice were Apoe −/− and were progeny of at least 10 generations of C57BL/6 backcrosses.…”

Antiphospholipid antibodies induce thrombosis by PP2A activation via apoER2-Dab2-SHC1 complex formation in endothelium

“…An endothelial focus for preventive and therapeutic interventions against APS moving forward is potentially advantageous because strategies for endothelial cell phenotype manipulation in vivo continue to emerge, such as the use of helper-dependent adenovirus to provide stable transgene expression in the endothelium. 82,83 Nonviral technology using polymeric nanoparticles for endothelial siRNA delivery is also gaining momentum, 84,85 with endothelial-avid nanoparticles enabling multigene silencing already becoming feasible. 86 Now having greater understanding of the molecular underpinnings of the disease, and the cell type in which the processes likely primarily take place, there is hope that the development and application of mechanism-based therapies against APS-related thrombosis has become more feasible.…”