Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

Abstract

An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo

Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage

“…8 Our study shows that the protection provided by Bcl-2 to S/MAR-transfected hepatocytes was sufficient to achieve mitotic stability of a small proportion of S/MAR plasmid-containing hepatocytes in vivo. It has been previously reported that established cell clones containing S/MAR plasmids are found exclusively in euchromatin nuclear compartments of active transcription 18 and are associated with histone markers of active transcription (such as H3K4m3, H3K4m1 and H3K36m3) during most phases of the cell cycle.…”

“…8 For the experiments presented here we used this vector as a basis for the construction of a biscistronic expression cassette in which Bcl-2 is positioned as the first transcribed gene to provide high levels of Bcl-2 for protection against Fas-induced apoptosis, while expression of the second transgene luciferase permits evaluation of plasmid fate over time. The new S/MAR plasmid is called pBcLucA1 and its control without the S/MAR element is called pBcLucA1 Control.…”

“…For instance, after surgical removal of two-thirds of the liver and subsequent regeneration, transgene expression drops to 0.7% of the pre-hepatectomy level. 8 This loss of expression is not entirely unexpected, since in vitro studies also show a quick loss of S/MAR-vector pDNA within a week after transfection into dividing cells if no initial selection pressure is applied.…”

“…This replication-dependent assay was not performed on DNA from animals not treated with Jo2, as we expect passive episomal maintenance without selective pressure as previously published. 8 The Southern analysis (Figure 4) serves to compare the restriction pattern of pBcLucA1 in liver DNA isolated from the pBcLucA1+Jo2-treated animal group (lanes 7-9) with that from the pBcLucA1 Control group (lanes 10-12) and with the restriction pattern of pLucA1 in DNA isolated from the pLucA1+Jo2 group (lanes [13][14][15]. Lanes 1-6 show the control restrictions on pDNA prepared from bacterial cultures.…”

“…They are ubiquitously present and highly sequence-conserved in eukaryotic chromosomes and therefore provide less potential immunological risk. 6 In addition, S/MAR vectors are able to prevent epigenetic silencing 7,8 by insulating the transgene sequence from heterochromatinization, thus ensuring its maintenance in a transcriptionally active chromatin environment. A pDNA vector system called pEPI, containing the mammalian S/MAR elements of the human b-interferon gene cluster, has been shown to be stably retained as episome for several hundreds of generations after initial selective pressure in vitro in CHO-KI, 9 HeLa 10 and K562 cell lines.…”