A vitrectomy improves the transfection efficiency of adenoviral vector-mediated gene transfer to Müller cells

Abstract

The neural retina is a logical target of gene therapy for highest degree of gene expression was obtained by various ocular diseases. We developed a new gene delivmethods A (23.2% of total retinal area) and C (19.8%), ery method to the neural retina using an adenoviral vector while the lowest degree was obtained by method B (8.9%). with a high degree of gene transfection efficiency and lessThe inflammation was observed in all eyes and the value functional damage. An adenoviral vector bearing the lacZ of inflammation was quantified as the average inflammagene (AdCALacZ) was injected into the eyes of adult Wistory cell number in each microscopic field (cells per fields). tar rats after an SF 6 compression gas vitrectomy and left A moderate degree of inflammation was induced by for 30 min followed by washing with balanced salt solution methods B (28.3 cells per field) and C (27.5 cells per field) (BSS) (method A). Three other methods, comprising a simand a minimal degree of inflammation was induced by ple intravitreal injection (method B), an intravitreal injection method A (11.2 cells per field). We evaluated the retinal after an SF6 compression gas vitrectomy (method C) or a function by measuring an electroretinogram (ERG). The subretinal injection (method D), were also studied. The amplitudes of the ERG were depressed in all eyes treated gene expression was examined 6 days after the AdCAwith AdCALacZ. This depression was manifested most by LacZ injection. An immunohistochemical study for antimethods B and C, and least by method A. The deteriovimentin, antiglial fibrillary acidic protein and anti S100 ration in the ERG findings seemed to correlate with the protein antibodies showed the neural retinal cells (Mü ller intensity of inflammation. Our study showed that an intravicells) to be primarily transfected by methods A, B and C, treal injection with an adenoviral vector can transfer the while only a few cells were transfected by method D. The genes to the neural retinal cells and therefore a vitrectomy expression of ␤-galactosidase was visualized by X-gal and the subsequent removal of the adenoviral vector, can staining and the positive areas on each hemiflat mount thus significantly improve the transfection efficiency and specimen were measured by an image analyzer and then also reduce the degree of functional damage. were adopted as a value of gene transfer efficiency. The

Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1

“…Intravitreal injection of adenoviral vectors results in transduction of the corneal endothelial cells, iris, ciliary body and trabecular meshwork. 36,37 Transduction of retinal Muller cells has previously been reported after intravitreal injection of adenoviral vectors, [38][39][40] but is inefficient unless the injection is combined with a vitrectomy, 41 a procedure not performed in our study. We have not demonstrated tissue localisation of sFlt-1 by immunohistochemistry, but can speculate that a substantial proportion of soluble protein secreted by cells in the anterior segment and ciliary body may be directed by aqueous flow anteriorly through the pupil towards the trabecular meshwork.…”

Photocoagulation-Induced Retinal Gliosis Is Inhibited by Systemically Expressed Soluble TGF-β Receptor Type II via Adenovirus Mediated Gene Transfer

“…The gene transfer of dominant negative truncated-type receptor is another potential method for blocking TGF-␤ signaling, which was achieved by AdT␤-TR and inhibited retinal gliosis in the present study. However, the intravitreous injection of adenovirus vector still has a negative effect on the retinal function (Sakamoto et al, 1998). Regarding the application of this above-described modality on humans, the transient transgene expression by replicant-deficient adenovirus-mediated gene therapy for the blockade of TGF-␤ may be suitable for the limited therapeutic purpose of maintaining the retinal function for short periods after retinal injury or surgical recovery.…”

“…The rats were divided into three groups; each animal of the following groups received the intramuscular injection (right femoral muscle) of either AdLacZ or AdT␤-ExR (0.2 ml, 1 ϫ 10 Ϫ8 pfu) (n ϭ 6 in each group). To see the direct inhibition of TGF-␤ signaling, AdT␤-TR (0.05 ml, 0.25 ϫ 10 Ϫ8 pfu) was injected into the vitreous using 30-gauge needle by our previously described method (Sakamoto et al, 1998). Immediately after the injection, retinal PC was carried out at the following parameters: 8 spots per eye, Argon laser, a power of 50 to 100 mJ, an exposure duration of 50 msec, and a spot size of 100 m by our previously described method (Honda et al, 2000).…”

Simian immunodeficiency virus-based lentivirus vector for retinal gene transfer: a preclinical safety study in adult rats

“…The inflammatory response against vectors and/or transgene is an important factor affecting the retinal architecture and functions, as previously demonstrated by us and others in the use of adenovirus for retinal gene transfer. 9,23,24 An important finding obtained in this study is that the histological damage of the retina might occur after around days 60-90 when the functional damage assessed by ERGs had almost recovered (Table 1). In the early phase during functional impairment of retina (À30 days), both the transgene expression and the Figure 4 Dissecting macroscopic view (a, g) and histopathological findings (b, e, f, h) of rat eyes injected with a high titer (2.5 Â 10 8 TU/ml) of SIV-NLSlacZ at several time points after gene transfer.…”