A 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long-term expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

Abstract

Helper virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100bp fragments that each support long-term expression, and are contained within an ~320bp fragment located ~3kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100bp fragments contained 44 or 23bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42bp sequence, centered on the 23bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16bp maximum essential sequence. This 16bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.