In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer

White, Stephen J.; Papadakis, ED; Rogers, Chris; Johnson, Jason L.; Biessen, Erik A. L.; Newby, AC

doi:10.1038/sj.gt.3303058

Cited by 12 publications

(14 citation statements)

References 35 publications

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“…In conclusion, when compared to previous work that addresses either transcriptional control in HDAd or optimization of transgene expression in EC in vivo, 43,50 our results reveal new factors that influence the level and time course of transgene expression after in vivo transduction of EC with HDAd. These factors include susceptibility of the vector promoter to acute upregulation as well as feedback effects of transgene products (such as IL-10) on transgene expression.…”

Section: Discussionmentioning

confidence: 47%

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

Dronadula

Kwast

et al. 2017

Human Gene Therapy

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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.

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Section: Discussionmentioning

confidence: 47%

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

Dronadula

Kwast

et al. 2017

Human Gene Therapy

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“…The need for an EC-specific cassette that achieves expression levels in large vessel EC at least as high as those obtained with the CMV promoter is well stated in a recent article by White et al 16. We pursued this goal using rabbit IL-10 cDNA and genomic clones, murine ET-1 promoter and enhancer sequences, and a post-transcriptional regulatory element (PRE).…”

Section: Discussionmentioning

confidence: 99%

“…In several cases relative EC-specificity was achieved compared to viral or broadly active eukaryotic promoters such as phosphoglycerate kinase. However, EC-specificity was acquired at the cost of reliably lower transgene expression 12,14,16…”

Section: Discussionmentioning

confidence: 99%

“…However, far less attention has been devoted to maximizing transgene expression in EC of large blood vessels, in which transcriptional regulation is likely different than in smaller vessels 21. Many investigators use the cytomegalovirus (CMV) immediate early promoter to drive transgene expression in large vessel EC despite its lack of tissue-specificity and its susceptibility to attenuation and shutdown 16,22. The need for an EC-specific expression cassette than outperforms CMV in large vessel EC is well enunciated; however, attempts to construct such a cassette have not been successful 16…”

Section: Introductionmentioning

confidence: 99%

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Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

Dronadula

Flynn

et al. 2010

Gene Ther

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The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity, and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin-10) in large vessel endothelial cells (EC) at levels as high as obtained with the CMV immediate-early promoter, while retaining EC-specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC-specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely 4 copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large vessel EC, while minimizing systemic effects.

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“…Another challenge was in generating an EC-specific promoter with comparable efficiency as the CMV promoter. White et al [120] examined several novel Ad expression cassettes for EC-specific gene transfer with CMV, Tshort, ICAM-2, ICAM-1, FLT-1 promoters, respectively and found that LOX-1 promoter elements significantly increased reporter gene expression in carotid arteries compared to other promoters. The efficacy of these novel expression cassettes in large animal models have yet to be established.…”

Section: Tissue-specific Targetingmentioning

confidence: 99%

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

Pankajakshan¹,

Agrawal²

2013

Gene Therapy - Tools and Potential Applications

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In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer

Cited by 12 publications

References 35 publications

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

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

Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

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