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

Abstract

Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function in both health and disease. Genetically manipulating astrocytes is an essential tool in understanding and affecting their roles. Adeno-associated viruses (AAVs) enable rapid genetic manipulation; however, astrocyte specificity of AAVs can be limited, with high off-target expression in neurons and sparsely in endothelial cells. Here, we report the development of a cassette of four copies of six miRNA targeting sequences (4x6T) which triggers transgene degradation specifically in neurons and endothelial cells. When used in combination with the GfaABC1D promoter, 4x6T increases astrocytic specificity of Cre with a viral reporter from 99% in multiple serotypes in mice, and confers astrocyte specificity in two inducible forms of Cre; Dre; and reporters. We also present empty vectors to add 4x6T to other cargo, independently and in Cre/Dre-dependent forms. This toolbox of AAVs provides a way to rapidly manipulate astrocytes throughout the CNS, is compatible with different AAV serotypes, and demonstrates the efficacy of using multiplexed miRNA targeting sequences to decrease expression in multiple off-target cell populations simultaneously.