Abstract
Purpose: :
To evaluate the effects of Etoposide, a anticancer chemotherapeutant, on the rAAV2-mediated gene expression in retina both in vitro and in vivo.
Methods: :
The hRPE cell line, primarily cultured adult RPE and rat retina neural cells were infected by rAAV2-EGFP alone or with lower dosage of etoposide. EGFP expression was then observed and analyzed by using of inverted fluorescent microscopy, FACS, and Western blotting. The rAAV2-EGFP/rAAV2-Luc alone or with lower dosage of Etoposide was injected into subretinal space of either SD rats or Balb/c mice. EGFP expression in the living retina was monitored and photographed using fluorescent stereoscope. Bioluminescene imaging was performed to evaluate dynamics of transgene expression in vivo. This strategy, the combination of rAAV2 and Etoposide, was also investigated for the feasibility to augment the rescue of retinal degeneration. rAAV2-CNTF combined with or without Etoposide was injected into subretinal space of RCS rats at the age of 3 weeks. Histologic examination was carried out 5 weeks later to observe the protection of photoreceptor lose. The eyes were also examined for apoptosis using TUNEL in situ assay in normal SD and RCS rats.
Results: :
GFP expression was observed 12 h postinfection in the cells infected by rAAV2-EGFP with Etoposide, while cells infected by rAAV2-EGFP alone did not show EGFP expression until the 5th day. The percentage of EGFP positive cell and mean of EGFP fluorescence intensity detected by FACS increased from 13.5% and 2.75 in the rAAV2-EGFP infected cells to 91.7% and 103.5 in the rAAV2-EGFP with Etoposide infected cells at the7th day postinfection. Earlier onset of gene expression, stronger and prolonged fluorescene were observed in the rAAV2 with Etoposide injected eyes in comparison with rAAV2 injected eyes. And the RCS rats, received injection of rAAV2-CNTF and Etoposide, possessed four to five rows of photoreceptors whereas the control, received injection of rAAV2-CNTF alone, had only two to three rows of photoreceptors (p<0.01). There was no remarkable apoptosis and cytotoxity in SD and RCS rat retina.
Conclusions: :
The data from this study suggested that Etoposide could significantly augment rAAV2-mediated transgene expression in human, rat as well as mouse retinal cells. The use of Etoposide may enhance the utility of rAAV2-mediated transduction strategies for retinal gene therapy.
Keywords: retina • gene transfer/gene therapy • photoreceptors