Study On The Tropism Of RAAV6-S663L In Mouse Retina

Adeno-associated virus vector(AAV)is a gene therapy vector with high safety,low immunogenicity and broad infection.Because of the structural characteristics of eye tissue,AAV has become the most accepted gene delivery vector for retinal gene therapy.Muller cells play an important role in maintaining homeostasis and neuronal structural integrity,stability and it have been found to be involved in many retinopathy.The aim of this study is to identify a rAAV mutant which has increased tropism for Muller cell of mouse retina.Using amino acid mutagenesis,we created a rAAV6 capsid mutant,rAAV6-S663L.The packaged virus can effectively infect cells in vitro and express fluorescent proteins.In addition,rAAV6-S663L with Muller cell-specific promoters can be specifically expressed in Muller cells in vitro.In vivo imaging and retinal flat mount were employed to analyze the gene expression of rAAV6-S663L and wt rAAV6 in mouse retinal tissue.Retinal tissue cryosection,immunohistochemistry(IHC),Muller cell specific promoter controlled gene expression,and double AAV fluorescent protein co-expression were performed to determine the targeting of rAAV6-S663L for mouse retinal Muller cells.In vivo imaging,retinal flat mount and retinal tissue cryosection results showed that rAAV6-S663L and wt rAAV6 have different specific tropism in mouse retina and rAAV6-S663L is more preferentially targeting Muller cells.Muller cell specific promoter controlled gene expression experiments and IHC test confirmed that rAAV6-S663L has a higher tendency to infect Muller cells than wt rAAV6.Co-infection of mouse retina with one rAAV6-S663L expressing EGFP under the control of GFAP promoter and the other one expressing mCherry under the control of CMV promoter revealed co-expression of the two fluorescent proteins in Muller cells.The results confirmed that rAAV6-S663L has a higher tropism for Muller cells than wt rAAV6.Our findings could add a new useful tool for retinal disease gene therapy.