The Cell- and Tissue-Type Dependent Activity of the Mouse Aspa Promoter and its Proximal Enhancers

Aspartoacylase (ASPA) is the enzyme responsible for metabolizing n-acetylaspartate, and for which loss-of-function mutations are causal for Canavan disease (CD). Aspa is primarily expressed in oligodendrocytes, but it is also found throughout the body. However, Aspa regulation in most tissues remains poorly explored. We identified putative mouse Aspa regulatory elements in a 3.4 kb sequence upstream of the transcriptional start site using publicly available epigenetic datasets. The sequence includes two putative tissue-specific enhancer sequences, enh1 and enh2, two non-hypersensitive regions, and a proximal promoter. To define the functional architecture of the putative Aspa promoter, we generated a series of promoter mutants with each identified sequence element deleted singly or in series. Plasmid reporter assay experiments in six cell lines derived from distinct tissue types, and in vivo mouse experiments with Aspa promoter AAV9 vectors, allowed the quantification of promoter activity in multiple contexts. We find that in the plasmid and AAV context, enh1 acts as a broadly active silencer, and that enh2 acts as a broadly active enhancer. The Aspa promoter showed minimal activity in low-Aspa expressing cell-types and tissues, including neurons and heart, which has the potential to enhance the safety of future gene therapy treatments which may utilize this promoter. The large enh2 sequence was further found to contain two small and adjacent silencer and enhancer elements. Using dsDNA-affinity-mass spectrometry, we identified CUX1 as a candidate binding protein of the isolated silencer sequence. We tested the effect of CUX1 on Aspa promoter activity using motif deletion, overexpression, and siRNA knockdown strategies. These experiments suggest that CUX1 may act as a repressor of Aspa promoter activity. However, our experiments were unable to confirm that the interaction of CUX1 and the mouse Aspa promoter is direct. In summary, the mouse Aspa promoter recapitulates aspects of endogenous Aspa expression and may be useful in the future as an endogenously derived regulatory sequence for Canavan gene therapy.