An immune response after intraocular administration of an adenoviral vector containing a beta galactosidase reporter gene slows retinal degeneration in the rd mouse

Abstract

Background/aims: Retinal degenerations are a leading cause of blindness for which there are currently no effective treatments. This has stimulated interest in the investigation of gene therapy strategies for these diseases in a variety of animal models. A number of attempts have been made to prevent photoreceptor loss in the rd mouse model of retinal degeneration using adenoviral vectors containing either a copy of the missing functional gene or a gene encoding either a neurotrophic factor or an antiapoptotic factor. The authors have previously demonstrated that intraocular administration of an adenoviral vector containing a beta galactosidase gene (AV.LacZ) results in an immune response to viral gene products and beta galactosidase. Here the effect of the immune response on retinal degeneration is examined.

Methods: Juvenile rd mice were injected intravitreally with AV.LacZ and a proportion were depleted of either CD4+ or CD8+ T cells or both. Control animals were injected with PBS. The mice were sacrificed 10 and 20 days post-injection and their eyes embedded in paraffin wax and sectioned.

Results: 10 days after intravitreal injection of AV.LacZ, the outer nuclear layer contains an average of 2.5 rows compared with 1.5 in PBS injected animals (p<0.005). The protective effect of AV.LacZ is negated by immune suppression and does not extend beyond 20 days.

Conclusion: An immune response to vector and transgene products is able to slow degeneration in the rd mouse. This phenomenon should be taken into account when analysing the degeneration in the rd mouse following gene transfer.

Figure 1

Eyes were sectioned to the level of optic nerve head (ONH) and only sections taken close to either side of the ONH were evaluated to ensure that retinas of normally equivalent thicknesses were compared. A 5 µm paraffin wax section counterstained with haematoxylin and eosin (10× objective) taken from 22 day old rd mouse 10 days after intravitreal injection of 1 µl of PBS.

Figure 2

Examples of sections taken close to the ONH region of 22 day old rd mice 10 days after intravitreal injection of either (A) 1 µl of PBS or (B) 1 µl of AV.LacZ (1×10⁶ pfu). The 5 µm paraffin sections were counterstained with haematoxylin and eosin (40× objective). Eyes injected with PBS had between one and two rows of photoreceptors in the outer nuclear layer (ONL) whereas in some eyes injected with AV.Lac Z there were between three and four rows of photoreceptors.

Figure 3

Comparison of rd ONL thickness in six groups of animals following intraocular injection (io) of either PBS or AV.LacZ with and without T cell ablation as a result of intraperitoneal injection (ip) of either PBS or monoclonal antibodies (mAb) specific for CD4 or CD8. The number of eyes in each treatment group (from 1 to 6) is 12, 6, 6, 8, 10, and 10 respectively. The scoring system was as follows: 0, a sparse row of photoreceptors; 1, a single row of photoreceptors; 2, between one and two rows of photoreceptors; 3, between two and three rows of photoreceptors, and so on. The data show the standard error of the mean ONL score. The difference in ONL thickness between treatment groups 1, 2, and 3 is not statistically significant. There are also no statistically significant differences in ONL thickness between treatment groups 4, 5, and 6. There is, however, a significant difference between groups 1 and 4 (p> 0.01); 1 and 5 (p< 0.005); 1 and 6 (p< 0.001); 2 and 4 (p< 0.05); 2 and 5 (p<0.05); 2 and 6 (p<0.005); 3 and 4 (p<0.05); 3 and 5 (p<0.05); 3 and 6 (p<0.005).