The Extragenic Spacer Length Between the 5′ and 3′ Ends of the Transgene Expression Cassette Affects Transgene Silencing From Plasmid-based Vectors

Abstract

In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5' and 3' ends of the transgene expression cassette and determined the expression profiles using two different reporter expression cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3'-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the expression cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.

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“…[19][20][21] Thus, use of these vectors presents an opportunity to examine the effects of greatly increasing antigen expression without necessarily modifying the innate immune response to vaccination as in electroporation or similar approaches. [22][23][24] Recently, DNA minicircles were reported to elicit greater frequencies of antigen-specific CD8 C T cells when compared to conventional plasmid immunization, and minicircle immunization was able to better protect against infection by an intracellular bacterium.…”

Human Induced Pluripotent Stem Cells as a Platform for Personalized and Precision Cardiovascular Medicine

“…Nevertheless, the reprogramming efficiency achieved using nonreplicating vectors is relatively low and requires multiple transfections of the target cells (114,209). Potential explanations for this phenomenon include the low transfection efficiencies of large polycistronic plasmids and enhanced transgene silencing mechanisms operating on plasmid-based vectors in mammalian cells (167).…”

Nonviral Gene Therapy—The Challenge of Mobilizing DNA

“…This indicates that plasmid silencing is not due to aberrant mRNA processing and that transcriptional blockage is enforced through plasmid nucleosome formation and subsequent heterochromatin formation. Interestingly, injections into mouse liver of minicircles with various lengths of extragenic spacer sequences have indicated that the length and not the sequence or origin of this extragenic spacer is the key determinant, at least in this tissue, for transcriptional silencing of episomal transgene expression cassettes (Lu et al 2012).…”