Volume 20, Issue 9, Pages 1713-1723 (September 2012) Gene Transfer to the CNS Is Efficacious in Immune-primed Mice Harboring Physiologically Relevant Titers of Anti-AAV Antibodies  Christopher M Treleaven, Thomas J Tamsett, Jie Bu, Jonathan A Fidler, S Pablo Sardi, Gregory D Hurlbut, Lisa A Woodworth, Seng H Cheng, Marco A Passini, Lamya S Shihabuddin, James C Dodge  Molecular Therapy  Volume 20, Issue 9, Pages 1713-1723 (September 2012) DOI: 10.1038/mt.2012.114 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Anti-AAV antibody titers in human serum. (a) Total and neutralizing anti-AAV antibody titers in human serum for serotype 2/2. (b) Relative distribution of total and neutralizing anti-AAV antibody titers in human serum for serotype 2/2. (c) Total and neutralizing anti-AAV antibody titers in human serum for serotype 2/5. (d) Relative distribution of total and neutralizing anti-AAV antibody titers in human serum for serotype 2/5. Neutralizing anti-AAV antibody titers ≤25 were below the limit of detection. AAV, adeno-associated virus. Molecular Therapy 2012 20, 1713-1723DOI: (10.1038/mt.2012.114) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Effect of high circulating total anti-AAV2/5 antibody titers on AAV2/5-mediated transgene expression within the mouse CNS after intraparenchymal (IP) or intracerebroventricular (ICV) vector delivery. Pre-sx = before stereotaxic surgery. Post-sx = after stereotaxic surgery. (a) Serum and (b) brain total anti-AAV antibody levels in naive and immune-primed (intravascular pre-exposure to AAV2/5-βgal) mice versus untreated mice. Human insulin-like growth factor-1 (hIGF-1) levels in the (c) injected and (d) noninjected hemispheres of AAV nave and immune-primed mice after either IP or ICV injection of AAV2/5-hIGF-1 into the brain. Columns not connected by the same letter are significantly (P < 0.01) different from each other. ND = antibody titer (as shown in b) or IGF-1 protein levels (as shown in c and d) were below the limit of detection for the assay. AAV, adeno-associated virus; CNS, central nervous system. Molecular Therapy 2012 20, 1713-1723DOI: (10.1038/mt.2012.114) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Total serum anti-AAV2/5 antibody titers detected in mice following intravascular injection of AAV2/5-βgal at various doses. AAV, adeno-associated virus; Ab, antibody. Molecular Therapy 2012 20, 1713-1723DOI: (10.1038/mt.2012.114) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Effect of lower circulating levels of total anti-AAV antibody titers on AAV-mediated gene expression within the mouse CNS after intraparenchymal (IP) or intracerebroventricular (ICV) delivery. Pre-sx = before stereotaxic surgery. Post-sx = after stereotaxic surgery. (a) Serum and (b) brain total anti-AAV antibody levels in naive and immune-primed (by intravascular administration of Gamunex) mice versus untreated mice. Human insulin-like growth factor-1 (hIGF-1) levels in the (c) injected and (d) noninjected hemispheres of naive and immune-primed mice following CNS delivery of AAV2/2-hIGF-1 or AAV2/5-hIGF-1. Columns not connected by the same letter are significantly (P < 0.01) different from each other. ND = antibody titer (as showin in b) or IGF-1 protein levels (as shown in c and d) were below the limit of detection for the assay. AAV, adeno-associated virus; CNS, central nervous system. Molecular Therapy 2012 20, 1713-1723DOI: (10.1038/mt.2012.114) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Glial cell staining was not enhanced in mice with pre-existing immunity to AAV. GFAP (marker of astrocytes) and IBA1 (marker of microlglia) staining in naive and immune-primed mice following intraparenchymal or intracerebroventricular injection of AAV-hIGF-1. Brain sections were analyzed from mice killed at 5 and 30 days after AAV vector delivery to the CNS. Inset is higher magnification (×20) of area marked by *. Bar = 100 µm, for inset bar = 50 µm. AAV, adeno-associated virus; CNS, central nervous system; GFAP, glial fibrillary acidic protein; hIGF-1, human insulin-like growth factor-1; IBA1, ionized calcium-binding adaptor molecule 1. Molecular Therapy 2012 20, 1713-1723DOI: (10.1038/mt.2012.114) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Neuroinflammation was not enhanced in mice with pre-existing immunity to AAV. CD3 (T-lymphocyte marker) and H&E staining in naive and immune-primed mice after intraparenchymal or intracerebroventricular injection of AAV-hIGF-1. Brain sections were analyzed from mice killed at 5 and 30 days after AAV vector delivery to the CNS. Inset is higher magnification (×20) of area marked by *. Bar = 100 µm, for inset bar = 50 µm. AAV, adeno-associated virus; CNS, central nervous system; H&E, hematoxylin and eosin; hIGF-1, human insulin-like growth factor-1. Molecular Therapy 2012 20, 1713-1723DOI: (10.1038/mt.2012.114) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Pre-existing anti-AAV2/1 capsid antibodies did not interfere with hASM brain expression and correction of lipid storage. (a) High levels of anti-AAV2/1 capsid antibodies were detected in systemic circulation in ASMKO mice (at 24 weeks of age) that were pretreated with AAV2/1-CBA-Null at 12 weeks of age. Intracranial injection of AAV2/1-hASM (at 24 weeks of age) led to detection of significant anti-AAV2/1 antibodies in serum 6 weeks after surgery (at 30 weeks of age). (b) Analysis showed similar levels of hASM expression in the CNS of AAV2/1-hASM–treated animals in both the saline-pretreated and AAV2/1-CBA-Null–treated cohorts. A similar reduction in (c) sphingomyelin and (d) cholesterol was observed in both the saline-pretreated and AAV2/1-CBA-Null–treated cohorts. ***P < 0.01. Shown are the (e–g) in situ distributions of hASM enzyme and (h–k) cholesterol storage in the brains of (e,h) untreated ASMKO, (f,i) saline-pretreated ASMKO, (g,j) AAV2/1-pretreated ASMKO, and (k) untreated WT mice at 6 weeks postinjection of AAV2/1-hASM. AAV, adeno-associated virus; ASMKO, acid sphingomyelinase knockout; CNS, central nervous system; WT, wild-type. Molecular Therapy 2012 20, 1713-1723DOI: (10.1038/mt.2012.114) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions