The distribution, concentration, and toxicity of EGFP in retinal cells after genomic or somatic (virus-mediated) gene transfer

PURPOSE: The concentration of enhanced green fluorescent protein (EGFP) in individual photoreceptor cells of live mouse retina was quantified and correlated with physiological measurements of cell function. METHODS: EGFP protein levels in the retinas of mice injected subretinally by either one of two serotypes of adeno-associated virus (AAV; AAV2/5.CMV.EGFP; AAV2/2.CMV.EGFP) were quantified with a photon-counting confocal laser scanning microscope and compared with those of transgenic mice whose retinas expressed EGFP under the beta-actin (p beta Act) or human L/M-cone opsin (pLMCOps) promoter. Single-cell suction pipette recordings of single rods and whole-field electroretinograms (ERGs) were performed to assess retinal cell function. RESULTS: The highest levels of EGFP (680 mu M) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of p beta Act.EGFP mice contained 270 mu M EGFP, while their bipolars had 440 mu M. The cones of pLMCOps.EGFP mice expressed 60 mu M protein. The amplitudes of the major components of ERGs were within the normal range for all transgenic animals examined, and single cell recordings from living p beta Act.EGFP rods were indistinguishable from those of controls. CONCLUSIONS: EGFP levels in individual cells of live mouse retinas can be quantified, so that the efficacy of gene transfer methods can be quantified. Concentrations of several hundred mu M are not deleterious to normal function of photoreceptors and bipolar cells. This approach can also be used to quantify levels of biologically active EGFP fusion proteins.