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E. Sugano, H. Tomita, H. Isago, H. Yawo, T. Ishizuka, M. Tamai; Immune Responses to Adeno Associated Virus Type2 Encoding Channelrhodopsin-2 in Genetically Blind Rat Model for Gene Therapy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3008. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies reported that the transduction of channelrhodopsin-2(ChR2) which is derived from Chlamydomonas to retinal ganglion cells (RGCs) restored visual response in aged dystrophic Royal College of Surgeons (RCS) rats. In this study, we investigated immunological responses against the vector and the transgene during the long-term transgene expression.
Adeno-associated virus(AAV) vector encoding ChR2 gene was intravitreally injected into both eyes of RCS rats. Peripheral blood was collected pre- and post- the operation. Lymphocyte was separated by lysing blood cells and the number of leukocyte was counted. T cells were analyzed by a flowcytometer to assess the inflammation and the rejection. ChR2 protein expression in the retina was investigated by the observation of the fusion protein, venus in retinal whole mount specimen. Visually evoked potentials (VEPs) were recorded from visual cortex of the RCS rats by the flash of a blue light-emitting diode (LED).
After an year of ChR2 gene transfer, the ChR2 expression in retina and the recovered VEPs were investigated. There was no decrease of the protein expression and recorded amplitudes of VEPs. The number of leukocytes increased 2 weeks after the gene transfer. However, at one month of post-operation, the number of leukocytes was dropped to pre-operation level. As an inflammation maker, we investigated the ratio of CD4, CD8 and CD25 expression of T cells. There was no significant difference of the ratio during the experimental periods (1-year).
rAAV2 vector administration to retina appears well tolerated. The increase of leukocytes might reveal some immune responses induced at the early stage of the AAV integration. But expression and function of ChR2 was not rejected by the host immune response mechanism during 1 year of observation. These data demonstrated that our approach, ChR2 transgene to retina with AAV vector, is reasonable for long-term gene therapy.
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