407. A Single Unilateral Injection of AAV1-ASA and AAV1-FGE Vectors into the Hippocampus Results in Bilateral Expression and Widespread Distribution of ASA and Prevention of Sulfatide Storage in the Whole Brain of MLD Model Mice

Abstract

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). The disease is characterized by progressive demyelination and widespread deposition of sulfatide in both the central and peripheral nervous systems. Therefore, direct injection of viral vector through the blood brain barrier is a possible approach for gene therapy of MLD. Lysosomal enzymes including ASA are known to be secreted and recaptured by surrounding cells through the mannose-6 phosphate receptor mediated pathway. Despite of this unique property, to treat the whole brain, it is likely that a large number of vector particles should be repeatedly injected into multiple sites. High efficient gene transfer and expression are critical for direct gene therapy of the brain of MLD. We have previously reported that co-expression of formylglycine generating enzyme (FGE), which is a post-translational modifying enzyme essential for activating all sulfatases, is essential for over-expression of functional ASA both in vitro and in vivo. In this study, we examined the utility of FGE co-expression in AAV1 mediated gene therapy of ASA knockout (MLD) mice. A preliminary experiment showed that stereotactic injection of AAV1-GFP into the CA3 region of the hippocampus resulted in strong GFP expression in both ipsilateral and contralateral hippocampi, suggesting that AAV1 vector is efficiently transported through axons to distal cell bodies. In a treatment experiment, AAV1-ASA alone or AAV1- ASA plus AAV1-FGE (total 7.5 |[times]| 109particles) were injected into the right hippocampus CA3 region of 8 month-old ASA knockout mice. The treated mice were analyzed 7 months after injection. Colorimetric assay of brain homogenates showed that injection of AAV1-ASA alone increased ASA activity in the injected hemisphere along with a slight increase in ASA in the non-injected hemisphere. Co-introduction of AAV1-ASA and AAV1-FGE significantly increased ASA activity in both sides. Widespread distribution of ASA was also confirmed by immunohistochemical analysis using anti-ASA antibody. Alcian blue staining and chemical analysis showed that sulfatide in the injected hemisphere was decreased after AAV1- ASA injection. Remarkably, reduction of sulfatide in the whole brain was observed when AAV1-ASA and AAV1-FGE were co-introduced. Rotarod test and walking pattern revealed significant improvement of neurological functions. These results indicate that AAV1 mediated expression of ASA and FGE is highly efficient for long-term expression and secretion of functional ASA in the brain. Unexpectedly, ASA could be distributed to the brain areas distant from the site of gene transfer by axonal transfer and diffusion. Extensive distribution of ASA in the brain may rationalize direct gene therapy of MLD.