Stable and Efficient Lentiviral Gene Transfer of eGFP Under the Control of Novel Cone- and Rod-Specific Promoters

Abstract

Purpose: : Lentiviral vectors are considered an attractive alternative to rAAV for gene therapy protocols targeting photoreceptor cells because they have a cloning capacity up to 10 kb, cause a weak or absent inflammatory response, and express transgenes rapidly. Additionally, they transduce retinal non-dividing cells readily with high efficiency and provide stable and long-term gene expression in vivo. Here, we investigate the expression of the enhanced green fluorescent protein (eGFP) after subretinal injections of lentiviral constructs under the control of unspecific as well as cone- and rod-specific promoters.

Methods: : Constructs for the expression of eGFP under the transcriptional control of the CMV, Arrestin3, MWS, SWS, and Rho promoters were generated. EGFP expression was analyzed using confocal SLO and OCT immediately following subretinal injection and at three subsequent time points. Results were confirmed by immunohistochemistry.

Results: : The novel promoter constructs resulted in stable and efficient expression of eGFP in cone and rod photoreceptors. Fluorescence was detectable up to 4 months post injection using both ubiquitous and specific promoters, suggesting an integration of the plasmids into the genome. Transgene expression under the control of MWS/ SWS promoters was observed only if the injection sites were dorsal/ ventral, in agreement with the natural distribution of MWS and SWS cones. SLO/ OCT imaging showed a confined retinal detachment immediately following subretinal injection, that resolved within the first week, indicating that the procedure caused only minor defects.

Conclusions: : Our data show stable long term expression of the transgene under the control of cone- and rod-specific promoters, supporting the view that lentiviruses are a powerful tool for vector transport into retinal cells including photoreceptors. Consequently, lentiviral vectors can be expected to play an important role in future treatment strategies of retinal dystrophies.

Keywords: gene transfer/gene therapy • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • retina