Sustained Gene Expression in the Retina by Improved Episomal Vectors

Abstract

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.

Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells

“…The NGF concentration was significantly increased in pEGFP-C1-MAR-NGF, compared to that of CHO cells transfected with pEGFP-C1-NGF. These results are in agreement with those of a previous study (Calado et al, 2014). RT-PCR analysis indicated a significantly increased target NGF mRNA expression in CHO cells transfected with pEGFP-C1-MAR-NGF, compared to that in CHO cells transfected with the pEGFP-C1-NGF or pEGFP-C1 vectors.…”

“…MAR contains some characteristic sequence components, including a T-box (TTTTATTTTT), an A-box (AATAAAAA/CAA), an autonomous replicating DNA sequence, and fruit fly topoisomerase II recognition sites (Ehrhardt et al, 2008;Mucller and Flotte, 2008). The MAR also mediates the attachment of vectors to host cells (Chiaretti et al, 2008;Zheng et al, 2010;Calado et al, 2014).…”

Non-viral therapeutic approaches to ocular diseases: An overview and future directions

“…For example, a newer version of pEPI was constructed with a CpG-depleted backbone, called pEPito (171). Experiments have shown that pEPito can drive stable transgene expression at higher levels and for a longer time period than its successor pEPI in vitro and in vivo especially in the retina (171, 172). This has the added benefit of a minimized inflammatory response (173), which is often associated with typical CpG patterns in bacterial backbones (174).…”

“…Inclusion of this enhancer can be used to improve the expression of normally weak but tissue-specific promoters without compromising the specificity of the promoter (198), and has been implemented in the eye with the RPE65 promoter (172). This enhancer effect has also been demonstrated for regions from other promoters such as the photoreceptor-specific interphotoreceptor retinoid binding protein (IRBP), even though the effect is much less potent than in the case of CMV (65).…”

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells

“…The first nonviral and episomal plasmid vector pEPI based on the matrix attachment region (MAR), which can replicate autonomously with low copy numbers in all cells tested, was developed by Piechaczek et al [7]. However, the pEPI vector has several limitations, such as low copy number, unstable expression and low expression level [8,9].…”