Abstract
Adeno-associated virus (AAV) vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb) were obtained by progressively deleting the original 2.0-kb promoter from the 5’ end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength) and 0.2-kb (70% astrocyte specificity) promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity.
Highlights
Astrocytes express various receptors and transporters for neurotransmitters and are actively involved in neuronal processing by modulating local synaptic functions: impairment of astrocytes affects the synaptic function, leading to associated behavioral defects
Previous transgenic studies have shown that the 2.2-kb human GFAP promoter extending 2.2 kb upstream of the RNA start site serves as an astrocyte-specific promoter in the mouse brain [15]
We found that the whole cerebellum in mice treated with lentiviral vectors carrying the 2.0- or 1.6-kb promoter showed markedly brighter GFP fluorescence intensity than those treated with lentiviral vectors carrying the shorter cjGFAP promoters (Fig 2A–2F, upper panels)
Summary
Astrocytes express various receptors and transporters for neurotransmitters and are actively involved in neuronal processing by modulating local synaptic functions: impairment of astrocytes affects the synaptic function, leading to associated behavioral defects. Vectors derived from adeno-associated virus (AAV) and lentivirus are valuable for efficiently introducing transgenes into astrocytes [8,9,10]. AAV vectors have smaller diameters (~20 nm) than lentiviral vectors (~100 nm), and diffuse into the brain parenchyma, leading to transduction across large areas of brain tissue [8]. While lentiviral vectors can accommodate a transgene as large as 8 kb, AAV vectors have a capacity of only 4.7 kb, including a promoter sequence [11]. A compact astrocyte-specific promoter is valuable for AAV vector-mediated transgene expression
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