Canavan disease (CD) is a rare childhood leukodystrophy caused by autosomal recessive mutations in the aspartoacylase (ASPA) gene. Deficiency of ASPA in Canavan patients leads to the accumulation of N- Acetyl-Aspartic Acid (NAA), resulting in swelling and spongy degeneration of white matter in the brain. The clinical manifestations of this fatal disease include psychomotor retardation, muscular hypotonia, macrocephaly, head lag, seizures, and early death. Synthesis of NAA is carried out in the mitochondria of neurons by N-acetyltransferase-8-like (NAT8L) and hydrolyzed in oligodendrocytes by ASPA. Gene replacement therapy for ASPA deficiency is currently the most promising strategy for treating CD. Notably, we have recently achieved full therapeutic correction of the Canavan phenotype in the Aspa knockout (CD-KO) mouse model. A single intravenous injection of recombinant adeno-associated virus packaged with the human ASPA transgene (rAAV-hASPA) at early ages completely resolves neuropathology, resulting in treated animals that outperform wild-types in motor function tests. However, based on strong preliminary evidence, we now hypothesize that the CD phenotype presents a secondary etiology related to metabolism dysfunction. In addition, we recently revealed that overexpression of ASPA in wild type cells in vitro resulted in abnormal mitochondrial shape and function. These findings necessitate further preclinical investigations that focus on: 1) characterizing the possible toxicity of ASPA overexpression in cell types of the CNS and peripheral organs, 2) developing ASPA regulatory cassette(s) that can mimic endogenous physiological levels of ASPA to circumvent adverse effects that may exist due to treatment, and 3) determining the physiological and behavioral effects of ASPA overexpression using a clinically relevant non-human primate model. Our new research strategy now builds on our current promising progress and advances our goals for a safe and effective gene therapy for CD.
Canavan disease (CD) is a rare, childhood-lethal neurodegenerative disorder caused by autosomal recessive mutations in the aspartoacylase gene (ASPA). Recombinant adeno-associated virus (rAAV)-based ASPA gene replacement therapy is currently the most promising strategy for the treatment of CD. We have shown in preclinical studies that treating CD mouse models at neonatal stages with gene therapy can fully cure neurological defects, restore psychomotor function, and extend life. Although, treatments of animals can normalize inborn metabolic error and mitigate neuropathologies, preliminary in vitro analysis suggests that ASPA overexpression may cause dysregulation of cellular energy homeostasis. Here, we propose to investigate whether long-term expression of ASPA delivered by rAAV in both mouse and non-human primate models can lead to unexpected adverse outcomes. In addition, we aim to reduce potential toxicity by reengineering the vector regulatory cassette so that it carries native elements of the ASPA promoter in order to mimic normal ASPA expression. Knowledge gained from our new aims will extend the clinical applicability of rAAV-hASPA to safely and effectively treat CD in patients.
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