A MicroRNA124 Target Sequence Restores Astrocyte Specificity of gfaABC1D-Driven Transgene Expression in AAV-Mediated Gene Transfer

Grit Taschenberger; Julia Tereshchenko; Sebastian Kügler

doi:10.1016/j.omtn.2017.03.009

A MicroRNA124 Target Sequence Restores Astrocyte Specificity of gfaABC₁D-Driven Transgene Expression in AAV-Mediated Gene Transfer

Mol Ther Nucleic Acids. 2017 Sep 15:8:13-25. doi: 10.1016/j.omtn.2017.03.009. Epub 2017 Apr 6.

Authors

Grit Taschenberger¹, Julia Tereshchenko², Sebastian Kügler³

Affiliations

¹ Center of Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Goettingen, Germany; Department of Neurology, University Medical Center Goettingen, Waldweg 33, 37073 Goettingen, Germany. Electronic address: gtasche@gwdg.de.
² Department of Neurology, University Medical Center Goettingen, Waldweg 33, 37073 Goettingen, Germany.
³ Center of Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Goettingen, Germany; Department of Neurology, University Medical Center Goettingen, Waldweg 33, 37073 Goettingen, Germany.

Abstract

Experimentally restricting transgene expression exclusively to astrocytes has proven difficult. Using adeno-associated-virus-mediated gene transfer, we assessed two commonly used glial fibrillary acidic protein promoters: the full-length version gfa2 (2,210-bp human glial fibrillary acidic protein [GFAP] promoter) and the truncated variant gfaABC₁D (681-bp GFAP promoter). The capacity to drive efficient, but also cell-type specific, expression of the EGFP in astrocytes was tested both in vitro in rat primary cortical cultures as well as in vivo in the rat striatum. We observed an efficient, but not entirely astrocyte-specific, gfa2-driven reporter expression. gfaABC₁D exhibited a weaker activity, and most importantly, off-target, neuronal expression of the transgene occurred in a larger fraction of cells. Therefore, we explored the potential of a microRNA (miR)-specific target-sequence-based approach for abolishing off-target expression. When miR124 target sequences were incorporated into the 3' UTR, neuronal gene expression was effectively silenced. However, unexpectedly, the insertion of an additional sequence in the 3' UTR clearly diminished transgene expression. In conclusion, the gfaABC₁D promoter on its own is not sufficient to specifically target transgene expression to astrocytes and is not well suited for AAV-based gene targeting, even if short promoter sequences are required. The combination with a miR de-targeting sequence represents a promising experimental strategy that eliminates off-target, neuronal expression.

Keywords: GFAP; adeno-associated virus (AAV); astrocyte; gene therapy; gfa2; gfaABC(1)D; microRNA; promoter; targeting; transgene.