Onset of Immune Response is Caused by the Amount of Induced Protein, Not by the Viral Vehicle Type

Abstract

Abstract: : Purpose: Gene therapy in the retina could involve introduction of foreign proteins to the affected area. This project was designed to evaluate the immune response in the retina when a foreign protein is induced by viral transduction. Methods: EGFP expressing lentivirus and adeno-associated virus (rAAV) were introduced into the subretinal space of rabbit retina. The expression of EGFP as well as the fundus of the retina was followed by scanning laser ophthalmoscope (SLO). The amount of EGFP antibody present in the serum was monitored by testing the blood samples by ELISA. Results: EGFP expression could be observed after 3-4 days of introducing lentiviral vehicle into the subretinal space of a rabbit. When a large number of virions (≷10₇) are injected, vast majority of the cells in the targeted area show EGFP fluorescence with high intensity. EGFP expression remains unchanged for 1-2 weeks but then gradually disappears as the titer of antibody against EGFP increases. When a low number of virions (<10₅) are injected, only a few cells express the protein but no rejection could be observed. When a high titer rAAV harboring chicken beta-actin promoter driven EGFP (courtesy of Wm. Hauswirth) is introduced into the subretinal space, only a small number of cells start to fluoresce after 4-6 days. The intensity of the fluorescence increases for 2 weeks and then remains stable over the time of observation. Rejection is not seen. Conclusion: Even though the retina is considered an immune privileged site, over expression of foreign protein could provoke immune response and result in the rejection of targeted cells. Lentivirus prefers RPE cells to photoreceptors whereas rAAV infects PRs more efficiently. When RPE cells produce large amount of foreign protein, those cells will be recognized by the immune system and rejected causing severe retinal damage. This could be problematic when applying gene therapy. Whether immune suppression would overcome this phenomenon is under investigation. It can be suggested that rAAV transduction of EGFP is not rejected because of a low number of infected cells and / or the preferred infectivity of PR over RPE, since PR does not present surface antigens. The results confirm that the rejection is due to the amount of protein produced and not the viral vehicle type.

Keywords: 419 gene transfer/gene therapy • 554 retina • 433 immune tolerance/privilege