351. Efficacious Non-Oligodendrocyte Gene Therapy Suggests a New Dogma About CNS Compartmentalization of NAA Metabolism and Supports a Metabolic Sink Theory

Abstract

N-acetylaspartate (NAA) is the second most abundant amino acid derivative in the mammalian central nervous system (CNS). Although its physiologic function remains elusive, many CNS disorders have been associated with changes in NAA levels, e.g. Alzheimer's disease, bipolar disorder. The disease that is known to have direct connection to altered NAA metabolism is Canavan disease (CD); a leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene. The current understanding is that, physiologically, the ASPA enzyme hydrolyzes NAA into L-aspartate and acetate in oligodendrocytes. Consequently, it was postulated that Canavan gene therapy has to restore ASPA expression in oligodendrocytes. However, we hypothesized that NAA can move freely and its cell-type independent break-down ameliorates Canavan disease. We constructed several tissue/cell-specific expression cassettes limiting hASPA expression to either astrocytes, neurons, oligodendrocytes, liver, heart, or muscle. We opted for the Canavan disease knock-out (CD KO) mouse model because it shows early lethality at around post-natal day 28 and the severest disease phenotype of all Canavan mouse models available. To our surprise, mice expressing hASPA restricted to peripheral organs showed extended survival and normalization of the growth curve at later time points, suggesting a contribution of peripheral organs to the disease pathomechanism. Moreover, astrocyte restricted hASPA expression produced the strongest disease recovery matching the performance of wild-type (WT) mice. Thus, our data seem to support our oligodendrocytes independent NAA metabolic sink theory. In an attempt to verify this theory, we used lower dose rAAVhASPA for localized brain injections to demonstrate that localized T2 hyperintensity signal clearance on MRI was well correlated with reduction of NAA levels by MRS. In other words, the further away from the injection site, the higher the NAA levels, which supports the idea of drainage and hydrolytic activity of NAA towards the injection side. Currently, we are investigating this metabolic sink theory in more detail by creating a functional map of therapeutic gene transfer in the brain by mass spectrometry quantification of NAA, and vector genome and ASPA transcripts analyses in different anatomic regions. Overall, our data present evidence that ASPA expression does not have to be restored in oligodendrocytes in order to rescue lethality and Canavan disease phenotype, which is in congruence with the metabolic sink theory. Furthermore, this calls into question a common hypothesis that NAA breakdown in oligodendrocytes is the mainstay for myelination and myelin defects in Canavan disease and might even support the idea that other metabolic disorders could benefit from treatment under the premise of the metabolic sink theory.