Effect of Tissue-Specific Promoters and microRNA Recognition Elements on Stability of Transgene Expression After Hydrodynamic Naked Plasmid DNA Delivery

Abstract

Intravenous hydrodynamic injections into the liver and skeletal muscle have increased the efficacy of naked DNA delivery to a level that makes therapeutically relevant gene transfer attainable. Although there are no concerns about the immunogenicity of the delivered DNA itself, transgene products that are foreign to the host can trigger an immune response and hamper the therapeutic effect. Our goal was to determine whether and to what extent some known preventive measures are applicable to these delivery methods in order to achieve longterm expression of foreign proteins in immunocompetent mice. We designed plasmid DNA vectors that expressed a marker gene under the control of either a ubiquitous or a tissue-specific promoter. We also included microRNA (miR) target sites in the transcripts in order to silence expression in antigen-presenting cells (APCs). The constructs were delivered either into muscle or liver, using outbred ICR and inbred C57BL=6 mice. The data suggest that firefly luciferase, a potent immunogen, triggered a uniform immune response only in outbred ICR mice, and only when expressed from a ubiquitous promoter. This response could not be prevented by including APC-specific miR target sites in the transcript. In contrast, the probability of immune rejection in ICR mice could be significantly diminished by using tissue-specific promoters, and under these circumstances, the silencing of transgene expression in APCs did confer some benefits. After a single hydrodynamic injection, inbred mice did not reject luciferase under any of the tested conditions for at least 8 weeks. To test whether they became tolerized, they were challenged with a second boost of a cytomegalovirus promoter-driven luciferase construct. This triggered a strong immune response, suggesting that luciferase-reactive cells from the animals' T and B cell repertoire had not been eliminated. This secondary reaction could not be prevented by silencing expression in APCs. In conclusion, for the clinical application of hydrodynamic naked DNA delivery the use of tissue-specific promoters in combination with silencing expression in APCs will increase the probability of long-term expression, but the most desirable outcome, the establishment of transgene tolerance, appears unlikely to be achieved by any of these measures.

Effective Prevention of Liver Fibrosis by Liver-targeted Hydrodynamic Gene Delivery of Matrix Metalloproteinase-13 in a Rat Liver Fibrosis Model

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