A rAAV2/6 Mutant with Enhanced Targeting for Mouse Retinal Müller Cells

ABSTRACT

Purpose

Adeno-associated virus vector (AAV) is the most accepted gene delivery vector for retinal gene therapy. Müller cells play an important role in maintaining homeostasis and neuronal structural integrity, stability and it has been found to be involved in many retinopathies. The aim of this study is to identify a rAAV2/6 mutant which has increased tropism for Müller cell of the mouse retina.

Materials and Methods

Using amino acid mutagenesis, we created a rAAV2/6 capsid mutant, rAAV2/6-S663L. In vivo imaging and retinal flat mount were employed to analyze the gene expression of rAAV2/6-S663L and wt rAAV2/6 in mouse retinal tissue. Retinal tissue cryosection, immunohistochemistry (IHC), Müller cell-specific promoter-controlled gene expression, and double AAV fluorescent protein co-expression were performed to determine the targeting of rAAV2/6-S663L for mouse retinal Müller cells.

Results

In vivo imaging, retinal flat mount and retinal tissue cryosection results showed that rAAV2/6-S663L and wt rAAV2/6 have different specific tropisms in mouse retina and rAAV2/6-S663L is more preferentially targeting Müller cells. Müller cell-specific promoter-controlled gene expression experiments and IHC test confirmed that rAAV2/6-S663L has a higher tendency to infect Müller cells than wt rAAV2/6. Co-infection of the mouse retina with one rAAV2/6-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 Müller cells.

Conclusions

The results confirmed that rAAV2/6-S663L has a higher tropism for Müller cells than wt rAAV2/6. Our findings could add a new useful tool for retinal disease gene therapy.

KEYWORDS:

• Raav2/6
• rAAV2/6-S663L
• Müller cells
• tropism
• gene therapy

Acknowledgments

The authors would like to thank the reviewers for pointing out many mistakes in our manuscript and Joyce X. Zhang for her reading of our manuscript and correcting the errors. The authors are thankful to Zheng Wei and Dongxin Wang for their reading of the manuscript and helpful comments and suggestions.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Grant number 81570875); and the Jiangsu Province Science Fund for Distinguished Young Scholars (Grant number BK20060010); the National Natural Science Foundation of China [81570875]; the Jiangsu Province Science Fund for Distinguished Young Scholars [BK20060010].