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Cheng-Hui Lin, Candace Chan, Man-Ru Wu, Alexander Davis, Sui Wang; Identification of cis-regulatory elements that allow for AAV-based Müller glial-specific labeling and manipulation in the retina. Invest. Ophthalmol. Vis. Sci. 2021;62(8):355.
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© ARVO (1962-2015); The Authors (2016-present)
Müller glial cells (MG), the major type of glia in the retina, provide structural and functional support to retinal neurons, and play important roles in a variety of retinal diseases. Recent advances in cell reprogramming also suggest that regeneration of desired neurons from MG in mammalian retina may offer promising cues for retina diseases. To study the function of MG and stimulate their regeneration, developing the molecular tools that allow for easy access of MG in vivo in mammalian retina are of fundamental importance. Adeno-associated viruses (AAVs)-based tools that can specifically label MG have been developed. However, there are critical limitations. First, the commonly used GFAP promoter often responds to disease insults in MG, leading to uneven expression under control and diseases conditions. Second, AAVs that can direct gene manipulation via the Cre-LoxP system in MG are not available. The goal of this study is to overcome these limitations, and develop engineered AAVs that allow for MG-specific labeling and manipulation in mammalian retina.
We first searched for cis-regulatory elements (CREs) that could drive MG-specific expression and were unresponsive to disease insults. We then integrated these CRMs into AAV vectors to drive EGFP or Cre/CreER expression, and tested their specificity in vivo in mice. The neurotoxin NMDA was used to investigate whether these CRMs respond to injuries. We compared the specificity of the identified CRMs with the published GFAP promoter in this study.
We identified multiple novel CRMs that can direct expression specifically in MG in vivo in mice. When integrated into AAVs, one of these CRMs, namely MG1, was able to drive MG-specific expression, and its activity was not affected in the presence of NMDA. In addition, the AAV-MG1-CreER virus can activate the tdtomato reporter gene specifically in MG in a tamoxifen and Cre-dependent manner in Ai9 mice. More importantly, the MG1 CRM was only 93bp, which does not impact the packaging capacity, allowing for additional genes in AAVs.
We identified novel MG-specific cis-regulatory elements, and developed novel AAV tools that can specifically label and manipulate MG in an injury-independent manner in vivo. These tools can significantly benefit the study of MG function and regeneration in mammalian retina.
This is a 2021 ARVO Annual Meeting abstract.
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