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Stable In Vivo Transgene 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.

SUBMITTER: Dronadula N

PROVIDER: S-EPMC5367915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Joint trauma leads to post-traumatic inflammation with upregulation of inflammatory cytokines and degradative enzymes. If severe enough, this response can lead to irreversible post-traumatic osteoarthritis. Interleukin-10 (IL-10), a cytokine with potent anti-inflammatory effects, has been shown to have chondroprotective effects. A gene therapy approach using a vector to overexpress IL-10 in the joint represents a feasible method of delivering sustained high doses of IL-10 to post-traumatic joints. We hypothesized that an AAV5 vector overexpressing IL-10 would result in rapid and sustained IL-10 expression following direct intra-articular injection and that this increase would not be reflected in systemic circulation. In addition, we hypothesized that intra-articular AAV5-IL-10 injection would not induce a local inflammatory response. Twelve horses were assigned to either treatment (AAV5-IL-10-injected) or control (PBS-injected) groups. Middle carpal joints were injected with 1012 vector genomes/joint or phosphate-buffered saline (PBS) alone (3 mL). Serial synovial fluid samples were analyzed for inflammatory changes, IL-10 concentration, and vector genome copy number. Serum samples were also analyzed for IL-10 concentration and vector genome copy number. Synovial membrane was collected on day 84. Synovial fluid IL-10 was significantly increased within 48 h of AAV5-IL-10 injection and remained increased, compared to PBS-injected joints, until day 84. Serum IL-10 was not different between groups. Vector administration did not cause a significant synovial inflammatory response. Vector genomes were detectable in the plasma, synovial fluid, and synovial membrane of AAV5-IL-10-injected horses only. IL-10 has the potential to modulate the articular inflammatory response, thereby protecting cartilage from degradation and osteoarthritis. This study demonstrates the feasibility and efficiency of intra-articular AAV5-IL-10, and future studies investigating the chondroprotective effects of IL-10 in inflamed joints in vivo are warranted.

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Stable In Vivo Transgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10.

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