Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity

Glover et al. (2002). Mol Ther DOI: 10.1006/mthe.2002.0588 Citations: 124

Neuron-specific promoters were too large for viral vectors and expressed weakly. A cassette combined the compact 495-bp SYN1 promoter with an 800-bp WPRE element in an adenovirus. It raised EGFP expression with no loss of neuronal specificity for six weeks in vivo.

Parts used

hSyn PromoterWPREEGFPCMV Promoter

Key findings

  • A composite adenoviral expression cassette combining 495 bp of the human SYN1 (synapsin-1) promoter with 800 bp of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was constructed. In non-neuronal HeLa cells, SYN1-driven vectors produced no fluorescence above background regardless of WPRE addition, confirming complete neuronal specificity.

  • Addition of WPRE to the SYN1-EGFP adenoviral construct increased EGFP fluorescence approximately 3-fold by fluorometry and 2.2-fold by FACS in primary dissociated hippocampal cultures. Neuronal specificity was maintained across all MOIs and time points, demonstrating that WPRE enhances expression without compromising the cell-type restriction of the SYN1 promoter.

  • In vivo injection of SYN1-WPRE-EGFP adenoviral vectors demonstrated enhanced transgene expression in dentate-gyrus neurons with no loss of neuronal specificity for at least 6 weeks post-transfection. This supports use of the SYN1-WPRE cassette for long-term neuron-specific gene delivery at lower viral titers, potentially reducing immunogenic inflammatory responses associated with higher viral loads.

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Abstract

Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.