Dataset Information

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

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.

SUBMITTER: Taschenberger G

PROVIDER: S-EPMC5476465 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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A MicroRNA124 Target Sequence Restores Astrocyte Specificity of gfaABC<sub>1</sub>D-Driven Transgene Expression in AAV-Mediated Gene Transfer.

Taschenberger Grit G Tereshchenko Julia J Kügler Sebastian S

Molecular therapy. Nucleic acids 20170406

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 gfaABC<sub>1</sub>D (681-bp GFAP promoter). The capacity to drive efficient, but also cell-type specific, expression of the EGFP in astrocytes was tested both ...[more]

PMID: 28918015

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Project description:Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here, we investigated whether incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3'-untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that five copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and ?-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23-fold in the liver. However, gene expression in other tissues, including the heart was not inhibited. Similarly, we inserted four copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3'-UTR of the AAV-luciferase vector. We wished to see whether they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time, regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly because of promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that liver-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in nonhematopoietic tissues resulted from promoter shut-off.

Dataset Information

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

Publications

A MicroRNA124 Target Sequence Restores Astrocyte Specificity of gfaABC<sub>1</sub>D-Driven Transgene Expression in AAV-Mediated Gene Transfer.

Similar Datasets

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Dataset Information

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

Publications

A MicroRNA124 Target Sequence Restores Astrocyte Specificity of gfaABC<sub>1</sub>D-Driven Transgene Expression in AAV-Mediated Gene Transfer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

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