Development of Persistently Expressing Vectors for the Non-Viral Gene Therapy of Choroideremia

Abstract

Purpose: : Non-viral gene therapy is an attractive and promising alternative to a viral-based approach due to biosafety and low immunogenicity of plasmid-based vectors. A novel class of vectors employing scaffold/matrix attachment regions (S/MARs) was developed recently. S/MAR vectors provide stable and functional episomal maintenance, resistance to epigenetic silencing and have been shown to be capable of sustained expression in murine tissues. Choroideremia (CHM) is an X-linked chorioretinal degeneration, currently incurable, which is caused by the loss of function of the CHM/REP1 gene. The aim of our current investigation is to develop persistently expressing non-viral vectors for gene therapy treatment of CHM.

Methods: : We generated S/MAR based vectors that contained either CHM/REP1 cDNA or EGFP driven by the EFS (human elongation factor 1 short) promoter. AtT20 cell line and choroideremia fibroblasts were transfected with DNA constructs, expression of EGFP and CHM/REP1 was analysed by Western blot analysis and FACS (for EGFP only). Cationic polymer polyethylenimine (PEI) was used to condense vector DNA and the DNA:PEI complex was injected subretinally into mouse eyes. Only one eye was injected with the contralateral eye serving as a control. To follow expression within the same animal we used a luciferase S/MAR construct (pEPI-Luc), were expression of luciferase was measured by an in vivo bioimager.

Results: : pEOS-Rep1 plasmid provides expression of hREP1 after transfection of the mouse AtT20 cell line. Similarly, EGFP expression conferred by plasmid pEOS-EGFP was confirmed by FACS after transfection of the AtT20 cell line. We established permanent AtT20 pEOS-GFP and AtT20 pEOS-Rep1 cell lines. Stable maintenance of pEOS vectors was monitored during culturing AtT20 pEOS-GFP cells over a period of time with and without selective pressure. Expression levels of the transgene did not diminish after 5 weeks. DNA:PEI complexes were successfully delivered to the RPE by subretinal injections. Expression of the transgene (EGFP or luciferase) in the RPE was confirmed.

Conclusions: : We have developed an S/MAR vector that is expressing the CHM/REP1 protein and could be used as a therapeutic agent for the non-viral gene therapy treatment of CHM. Our experiments demonstrate that S/MAR vectors are delivered to the RPE and thus could be used for the non-viral treatment of the RPE defects in ocular diseases.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • retinal pigment epithelium