Abstract
Purpose: :
Non-viral vectors are attractive alternatives to viral gene delivery systems due to their low toxicity, relatively easy production, and great versatility. Recently, we developed a novel plasmid vector employing a scaffold/matrix attachment region (S/MAR) to provide functional episomal persistence within cells and clinically relevant levels of transgene expression, in vivo, without vector toxicity.We will provide evidence for the utility of S/MAR vectors in the eye.
Methods: :
We have employed our S/MAR vector technology for the development of novel non-viral persistently expressing vectors for Choroideremia (CHM) gene therapy. We have created a series of constructs with either the reporter genes EGFP and Luciferase encoded along with REP1, with various promoters, with and without an S/MAR element.
Results: :
We performed initial experiments to evaluate the expression of the transgenes in vitro in a range of cell lines and demonstrated that the S/MAR plasmids provide long-term transgene expression in vitro.We subsequently optimised a procedure for the delivery and expression of these non-viral constructs in vivo via subretinal injection of formulated DNA. We will present data showing the longitudinal transgene expression of constructs measured using a bioluminescence bioimager for over 12 months. Additionally, long term expression of transgenes were also observed by rtPCR and Western blot analysis of protein levels. We will also show that evidence for the persistent episomal maintenance of these vectors in the eye.We show that the S/MAR motif in these vectors is essential for providing long term expression and maintenance in the eye.We also demonstrate the lack of toxicity within the eye and show that fundus examinations as well as detailed histological examinations of retinal sections do not elicit an inflammatory response to our plasmids once subretinally injected in the eye.
Conclusions: :
In conclusion, an ideal non-viral vector for gene therapy should be non-toxic, have mitotic stability, allow non-integrative establishment and provide persistent therapeutic expression levels. The S/MAR-containing vector that we have developed and applied fulfils these requirements and provides proof of principle for a new class of vector systems for ocular gene therapy.
Keywords: gene transfer/gene therapy • retinal pigment epithelium