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.

FIG. 1.

Transfection efficiencies for transiently transfected D407 cells with the five different plasmids. Mean values are derived from four independent experiments and statistical significance was determined with one-way ANOVA followed by a post hoc Tukey Test. The statistical difference is indicated by a star (*) symbol (*p<0.05).

FIG. 2.

Brightfield (A) and fluorescence (B) microscopy of a stably transfected D407 cell line colony with pEPito-CMV-eGFP-BSD (1), pEPito-hCMV-eGFP-BSD (2), and pEPito-hCMV/RPE65-eGFP-BDS (3) 32 days post-transfection. BSD, blasticidin; CMV, cytomegalovirus immediate early promoter; hCMV, human CMV enhancer/human elongation factor 1 alpha promoter; eGFP, enhanced green fluorescent protein; RPE, retinal pigment epithelial. Color images available online at www.liebertpub.com/tea

FIG. 3.

Colony-forming efficiency of D407 cells selected with blasticidin, for each of the pEPito plasmids. Mean values are derived from three independent experiments and statistical significance was determined with one-way ANOVA followed by a post hoc Tukey Test. Bars labeled with * indicate statistical difference (p<0.05).

FIG. 4.

Flow cytometry of D407 colonies stably transfected with pEPito-hCMV (B, F), pEPito-CMV (C, G), and pEPito-hCMV/RPE65 (D, H). Upper panels represent cells after 2 months of selection with BSD, and lower panels correspond to cells after 3 months of selection. (A) and (E) are the control nontransfected cells.

FIG. 5.

Transversal sections of a mouse retina injected with pEPI-1- and pEPito-based vectors, sacrificed 32 days postinjection. * Indicates clusters of eGFP-expressing ganglion cells transfected with pEPito-hCMV (A) (magnification: 50×); (B) corresponds to an amplification of image (A) (magnification: 200×), pEPito-CMV (C) (magnification: 200×), pEPI-1 (D), and pEPito-hCMV/RPE65 (E). (F) A noninjected retina, without GFP expression. DAPI (blue) stains nuclei. GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer. Color images available online at www.liebertpub.com/tea