Enhanced brain distribution of modified aspartoacylase

Abstract

Canavan disease is a fatal neurological disorder caused by defects in the gene that produces the enzyme aspartoacylase. Enzyme replacement therapy can potentially be used to overcome these defects if a stable enzyme form that can gain access to the appropriate neural cells can be produced. Achieving the proper cellular targeting requires a modified form of aspartoacylase that can traverse the blood-brain barrier. A PEGylated form of aspartoacylase that shows dramatic enhancement in brain tissue access and distribution has been produced. While the mechanism of transport has not yet been established, this modified enzyme is significantly less immunogenic than unmodified aspartoacylase. These improved properties set the stage for more extensive enzyme replacement trials as a possible treatment strategy.

Keywords: Aspartoacylase; Blood–brain barrier; Canavan disease; Enzyme replacement therapy; PEGylation.

Fig. 1

SDS-PAGE of PEGylated aspartoacylase. The gels were run under identical conditions, and were stained by using: (A) Coomassie blue for proteins or (B) Barium-iodine for PEGs. Lanes 1-2 are duplicates of the reaction mixture of ASPA with methoxy-PEG 5 kDa. Lane 3 is the protein molecular weight standards in (A & B) and Lane 4 is the PEG molecular weight standards in (B).

Fig. 2

Brain imaging (hippocampus) after treatment with fluorescently-labeled aspartoacylase. Panel A: treatment with unmodified enzyme showing confinement primarily in the capillary regions, Panels B through E: treatment with PEGylated enzyme at increasing dosing levels, 0.8 mg/kg; 1.3 mg/kg; 2.0 mg/kg and 3.0 mg/kg, respectively, showing diffusion into the surrounding tissues.

Fig. 3

Fluorescence quantification of brain hippocampus enzyme levels after eliminating basal and outlier fluorescence as described in methods. These results are the averages of three brain slices from each of two animals, with the standard deviations shown in the error bars.

Fig. 4

Immune response in the hippocampus region of the brain after treatment with fluorescently-labeled aspartoacylase (ASPA), measured with two different biomarkers normalized to enzyme fluorescence intensity illustrated in Fig 3. Panel A: GFAP marker, Panel B: IBA marker. Each treatment condition was assessed from two animals per group with three brain sections examined per animal.