Develop an efficient and specific AAV-based labeling system for Muller glia in mice

Abstract

Reprogramming Müller glia (MG) into functional cells is considered a promising therapeutic strategy to treat ocular diseases and vision loss. However, current AAV-based system for MG-tracing was reported to have high leakage in recent studies. Here, we focused on reducing the leakage of AAV-based labeling systems and found that different AAV serotypes showed a range of efficiency and specificity in labeling MG, leading us to optimize a human GFAP-Cre reporter system packaged in the AAV9 serotype with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) removed. The leakage ratio of the AAV9-hGFAP-Cre-ΔWPRE decreased by an approximate 40-fold compared with the AAV9-hGFAP-Cre-WPRE labeling system. In addition, we validated the specificity of the AAV-ΔWPRE system for tracing MG reprogramming under Ptbp1-suppression and observed strict non-MG-conversion, similar to previous studies using genetic lineage tracking mouse models. Thus, the AAV9-hGFAP-Cre-ΔWPRE system showed high efficiency and specificity for MG labeling, providing a promising tool for tracing cell fate in vivo.

A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences

“…We also generated a vector using the smaller GfaABC1D promoter 15 (PHP.eB::GfaABC1D-Cre). As only low levels of Cre are necessary for recombination, we omitted a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), used to increase transgene expression, reasoning that this might decrease off-target transgene expression; a similar approach was recently used to improve targeting of Muller glia 16 . Viruses were delivered systemically using retroorbital administration into young adult (2-5 month) C57BL/6J mice.…”

Advances in the study of Müller glia reprogramming in mammals

“…Furthermore, this research has advanced our understanding of the high leakage issue in AAV-based systems for tracing Müller glia. For instance, Gao’s study introduced the AAV9-hGFAP-Cre-ΔWPRE system, which, when compared to the conventional AAV9-hGFAP-Cre-WPRE labeling system, demonstrates enhanced efficiency and specificity in Müller glia labeling ( Gao et al, 2022 ).…”

“…It also systematically ruled out the impact of virus titer on fluorescence leakage issues and expressed substantial skepticism regarding the assertion that “the Cre-loxP recombination may have created a higher barrier for cell conversion” This thorough examination raises significant doubts about the reliability of the original study. This kind of research has resulted in the development of a safer, more efficient, and highly specific labeling system for Müller cells, which offers a promising tool for in vivo tracing of cell fate ( Gao et al, 2022 ). To sum up, addressing these questions is crucial for comprehending the transition to reprogramming in mammalian Müller cells and holds potential for future clinical applications.…”

The Healthy and Diseased Retina Seen through Neuron–Glia Interactions

“…However, the demonstration of the lack of specificity of the alleged glial promoters raised concerns in some of these studies and highlighted the need for proper strategies for tracing the MG as the cell of origin in protocols for MG to neuron reprogramming [477][478][479][480]. Research groups are also searching for alternative or complementary tools for MG reprogramming, such as investigating ways to identify novel cell-type specific regulatory regions to drive gene expression [481], modifications in AAV-carried sequences testing modifications in AAV-carried sequences [482], or screening compounds to increase the neurogenic reprogramming of the MG [483]. A great advance was obtained in the last decades on the identification of the critical approaches necessary to obtain reliable information, which could work as a proof of principle for the investment in regenerative strategies for ocular diseases [484].…”