Volume 11, Issue 5, Pages 745-753 (May 2005) Restoration of aspartoacylase activity in CNS neurons does not ameliorate motor deficits and demyelination in a model of Canavan disease  Matthias Klugmann, Claudia B. Leichtlein, C. Wymond Symes, Tadao Serikawa, Deborah Young, Matthew J. During  Molecular Therapy  Volume 11, Issue 5, Pages 745-753 (May 2005) DOI: 10.1016/j.ymthe.2005.01.006 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Restoration of ASPA deficiency after multisite rAAV delivery. (A) Scheme of rAAV injection into the striatum (left) and thalamus (right). (B) Experimental time line. Shown is the type of intervention at a given age in weeks (wk). (C) Representative immunoblots showing endogenous ASPA and transgene expression: whole hemisphere week 5 brain lysates probed with (a) anti-ASPA sera, (b) anti-HA, or (c) anti-GFP. (D) ASPA expression levels in whole brain hemispheres at all three time points were quantified from immunoblot signals standardized to β-actin and presented relative to mean week 5 wild-type levels. (E) ASPA enzymatic activity in the same samples as used in (D). (F) Correlation between ASPA protein levels derived from (D) and enzyme activity from (E). The bold and thin trend lines represent correlation in wt and tmASPA, respectively. Bars represent mean values ± SEM of 4 rats. Wt, uninjected wild type; tmASPA, AAV-ASPA-injected tremor rat; tmGFP, AAV-GFP-injected tremor rat. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001. Molecular Therapy 2005 11, 745-753DOI: (10.1016/j.ymthe.2005.01.006) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Brain morphology after rAAVaspa expression. (A) HA immunohistochemistry in sagittal section of a tmASPA brain. (B) Magnification of (A) showing HA-positive fibers in the hind brain. Arrows point to axonal swellings. (C–F) Confocal overlays of HA immunohistochemistry in the striatum of tmASPA brains, in red, and (C) CAII, (D) MBP, (E) GFAP, or (F) NeuN in green. Ectopic ASPA is not expressed in the small cell bodies of oligodendrocytes labeled by CAII (C), in myelin (D), or in astrocytes (E) but is exclusively detected in NeuN-positive neurons (F, arrowheads). Note that >95% of neurons in (F) are transduced. (G, H) Histopathology in treated tm rats. The numbers of vacuoles (arrows) appear similar in HandE-stained thalamus of (G) tmASPA and (H) tmGFP brains. Shown are representative sections of n = 4 or 5 per group. Bars: 2 mm (A); 100 μm (B); 10 μm (C–F); 250 μm (G, H). Molecular Therapy 2005 11, 745-753DOI: (10.1016/j.ymthe.2005.01.006) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 NAA measurement and seizure analysis. (A) Representative NMR spectra of perchloric acid lysates from whole hemispheres of wt (left) and tmGFP (right). (B and C) NAA levels presented as a ratio with creatine (Cr). For week 5 and week 11 time points (B), individual hemispheres (n = 4) were analyzed. At week 26 (C), tissue slices surrounding the thalamic injection site were combined (n = 4) and tested. (D) Representative hippocampal EEG recordings of spontaneous seizure activity in week 20 rats (n = 6–8 per group). Calibration in (D) 50 mV vertical, 5 s horizontal. (E) Average seizure occurrence quantified over 15-min sessions. EEGs were recorded three times per week, from week 18 to week 22. (F) Mean seizure length for the entire 4-week period. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001. Molecular Therapy 2005 11, 745-753DOI: (10.1016/j.ymthe.2005.01.006) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Myelin content and motor function. (A) Myelin measurements at all three time points. Shown is the myelin content in whole hemispheres from four rats per group. (B) Open field analysis shows immobility of both injection groups. (C) Gait analysis. Mean anterior–posterior distance between left and right hind paw (stride length) and medial–lateral deviation of left front and back paw derived from partially overlapping prints (paw spread). (D) Rotarod test. Shown are mean latencies (5 trials per rat) to fall off the stationary rod (phase 1), the constantly rotating (3 rpm) rod (phase 2), or the accelerating rod (phase 3). The maximum duration of an individual trial was 2 min (phases 1 and 2) or 7 min (phase 3). n = 8 for (B–D). ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001. Molecular Therapy 2005 11, 745-753DOI: (10.1016/j.ymthe.2005.01.006) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions