Oligodendrocyte-focused rAAV gene therapy strategies for Canavan disease and Leukodystrophies

PROJECT SUMMARY Zolgensma, an FDA-approved gene therapy, employs recombinant adeno-associated virus (rAAV) to treat spinal muscular atrophy (SMA). However, toxicity and even death have been reported in patients, potentially due to widespread transgene expression caused by a constitutively active and ubiquitous promoter. Consequently, we have developed a second-generation therapy employing a cell type-specific promoter to enhance efficacy while mitigating toxicity. Similarly, Canavan disease (CD), a leukodystrophy linked to the aspartoacylase (ASPA) gene, is targeted with rAAV-based gene therapy using a constitutively active and ubiquitous promoter, which has generated off-target effects in mouse and human. To bolster safety and efficacy, we have devised a novel ASPA promoter derived from the endogenous ASPA regulatory elements to emulate endogenous ASPA expression. In addition, understanding the regulation of the ASPA promoter aims at further improving specificity and therapeutic efficacy of the novel ASPA promoter (Aim 2). While rAAV's efficacy in transducing neurons is well-documented—beneficial for disorders like SMA—the understanding of rAAV's transduction of oligodendrocytes, the target cells in CD, is sparse. These cells play pivotal roles in neurodegenerative diseases such as Leukodystrophies, multiple sclerosis, and Alzheimer’s; hence, understanding their transduction by rAAV is crucial and has the potential to benefit diseases with oligodendrocyte involvement. Furthermore, understanding the mechanism of AAV capsid-oligodendrocyte interaction could inform novel capsid design for oligodendrocyte targeting and, together with a transgene- specific promoter, improve specificity and safety of oligodendrocyte-targeted rAAV gene therapy for CD (Aim 1). In conclusion, the urgent need to comprehend oligodendrocyte transduction, along with the promising advancements in gene therapy, emphasizes the significance of our study. The outcomes will enhance treatment safety and efficacy and broaden the application of gene therapy to oligodendrocyte diseases currently considered challenging to treat.