Volume 13, Issue 3, Pages 538-547 (March 2006) CNS-directed AAV2-mediated gene therapy ameliorates functional deficits in a murine model of infantile neuronal ceroid lipofuscinosis  Megan A. Griffey, David Wozniak, Michael Wong, Ellen Bible, Kendra Johnson, Steven M. Rothman, Annie E. Wentz, Jonathan D. Cooper, Mark S. Sands  Molecular Therapy  Volume 13, Issue 3, Pages 538-547 (March 2006) DOI: 10.1016/j.ymthe.2005.11.008 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 The graph shows the regional differences in mean PPT1-specific activity at 7 months of age in INCL mice given no (filled squares, n = 4), two (n = 6), four (n = 10), or six (n = 7) ic injections of AAV2-PPT1 at birth. PPT1-specific activity was measured in homogenates from 10 serial 1-mm coronal brain sections. Sections 1 through 10 represent activity from anterior to posterior, respectively. Positive immunostaining (brown) for PPT1 is observed in the (A) cortex, (B) hippocampus, and (C) cerebellum of an INCL mouse given six ic injections of AAV2-PPT1. Higher magnifications of these areas are shown (D–F). The staining in each area appears to be limited primarily to cells with characteristic neuronal morphology (G–I). Molecular Therapy 2006 13, 538-547DOI: (10.1016/j.ymthe.2005.11.008) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 (A) INCL mice (−/−, n = 4) have a significant increase in the amount of autofluorescent storage material (AF) in the brain at 7 months of age compared to WT animals (+/+, n = 4). INCL mice given as few as two ic injections (n = 5) or as many as six ic injections (n = 5) of AAV2-PPT1 show a significant reduction in the level of autofluorescent storage material in several regions of the brain compared to untreated INCL mice. The injection site in the anterior cortex is identified on the horizontal axis, and all other distances reported are listed in reference to this site (see [25] for a diagram of the sampling sites). (B) There is a trend toward increased cortical volume in INCL animals receiving six ic injections of AAV2-PPT1; however, the mean was not significantly greater (P = 0.073) than that in untreated INCL mice. Autofluorescence and cortical volume measurements were performed on the same animals. (C) INCL mice (n = 26) have significantly reduced brain weights compared to normal (WT, n = 26) animals. Animals given four (n = 12) or six (n = 7) injections have significantly increased brain weights compared to untreated INCL animals. The brain weights of INCL animals given six injections of AAV2-PPT1 are significantly less than those of WT mice. Animals given two injections (n = 9) showed no significant increase in brain weight compared to untreated INCL mice. Molecular Therapy 2006 13, 538-547DOI: (10.1016/j.ymthe.2005.11.008) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 (A) INCL mice (−/−, n = 4) have a significant increase in the amount of autofluorescent storage material (AF) in the brain at 7 months of age compared to WT animals (+/+, n = 4). INCL mice given as few as two ic injections (n = 5) or as many as six ic injections (n = 5) of AAV2-PPT1 show a significant reduction in the level of autofluorescent storage material in several regions of the brain compared to untreated INCL mice. The injection site in the anterior cortex is identified on the horizontal axis, and all other distances reported are listed in reference to this site (see [25] for a diagram of the sampling sites). (B) There is a trend toward increased cortical volume in INCL animals receiving six ic injections of AAV2-PPT1; however, the mean was not significantly greater (P = 0.073) than that in untreated INCL mice. Autofluorescence and cortical volume measurements were performed on the same animals. (C) INCL mice (n = 26) have significantly reduced brain weights compared to normal (WT, n = 26) animals. Animals given four (n = 12) or six (n = 7) injections have significantly increased brain weights compared to untreated INCL animals. The brain weights of INCL animals given six injections of AAV2-PPT1 are significantly less than those of WT mice. Animals given two injections (n = 9) showed no significant increase in brain weight compared to untreated INCL mice. Molecular Therapy 2006 13, 538-547DOI: (10.1016/j.ymthe.2005.11.008) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 AAV2-PPT1-treated INCL mice show less neurodegeneration in several areas of the brain. (A–D) Low-magnification images show the degree of silver staining (black) in 7-month-old WT, INCL (untreated MUT), two-injection INCL, and four-injection INCL mice. INCL mice given four injections showed less neurodegeneration in the anterior commissure (ac, arrowhead) and the lateral olfactory tract (lo, arrowhead) compared to untreated or two-injection INCL mice. (E–H) Less neurodegeneration and cortical thinning are observed in the anterior cortex of INCL mice given either two or four injections of AAV2-PPT1. (I–L) Reduced neurodegeneration in the CA1 region of the hippocampus (CA1, between the arrowheads) of PPT1-deficient animals is observed only in INCL mice given four injections of AAV2-PPT1. Molecular Therapy 2006 13, 538-547DOI: (10.1016/j.ymthe.2005.11.008) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 INCL mice treated with four (filled circles, n = 24) or six (open triangles, n = 12) injections of AAV2-PPT1 have significantly (P < 0.05) improved performance compared to untreated INCL mice (filled squares, n = 31) in (A) the continuously rotating rotarod and (B) the accelerating rotarod. There is no statistically significant difference between the six-injection INCL group and the WT animals (open squares, n = 24) in the accelerating rotarod assay (B). Similar improvements were seen in both (C) the pole and (D) the ledge tests, except that the four-injection group (AAV-PPT4) was not significantly different from INCL mice (−/−) in the ledge test. There is no significant improvement in the performance of INCL mice receiving two injections (AAV-PPT2, n = 10) of virus in any behavioral assay. There is no significant improvement in performance in any of the treatment groups in either (E) the total ambulations or (F) the center entries measurements during the open-field testing. Molecular Therapy 2006 13, 538-547DOI: (10.1016/j.ymthe.2005.11.008) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 (A) A graded scale from 1 to 4 was developed to quantify subtle changes in interictal EEG patterns in INCL mice. (B) There was a significant improvement in the interictal pattern (grade 2.21) in INCL mice (AAV-INCL) that received four injections of AAV2-PPT1 compared to untreated and AAV2-GFP-treated (grade 2.84) mice combined (INCL/GFP). However, that score was significantly different from that of WT mice (grade 1.2). (C) The EEG pattern of an INCL mouse during an observable seizure is shown. (D) Although the average number of seizures in INCL animals receiving four injections appeared to decrease compared to untreated INCL and AAV2-GFP-treated mice, the difference was not significant. No WT animal had a seizure (0) during any recording interval. Molecular Therapy 2006 13, 538-547DOI: (10.1016/j.ymthe.2005.11.008) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 INCL mice given either four (n = 21) or six (n = 5) injections of AAV2-PPT1 did not show any statistically significant increase in life span compared to untreated INCL (n = 19) or AAV2-GFP-treated INCL (n = 15) mice. No WT mice (n = 11) died during the course of the 1-year study. Molecular Therapy 2006 13, 538-547DOI: (10.1016/j.ymthe.2005.11.008) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions