Volume 25, Issue 11, Pages 2526-2532 (November 2017) Fatal Meningitis in Swine after Intrathecal Administration of Adeno-associated Virus Expressing Syngeneic Interleukin-10  Mark D. Unger, Josef Pleticha, James E. Collins, Anibal G. Armien, Jennifer L. Brazzell, Laura K. Newman, Lukas F. Heilmann, Jodi A. Scholz, Timothy P. Maus, Andreas S. Beutler  Molecular Therapy  Volume 25, Issue 11, Pages 2526-2532 (November 2017) DOI: 10.1016/j.ymthe.2017.07.016 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 Neuropathology of Moribund Animals (A–F) Representative H&E-stained images of the cerebellum (A and B), forebrain (C), brainstem (D), and spinal cord (E and F). The meninges were diffusely expanded by lymphocytic infiltrates (A–C and F), vascular congestion (arrowhead; A), and lymphatic distension (plus sign; C). Meningeal infiltration extended along the Virchow-Robin space forming perivascular cuffs (asterisk; D and E) and into adjacent gray matter with mild gliosis (arrows; C). Meningeal infiltrates were predominately lymphocytic with mild numbers of eosinophils in cerebellar meninges (B) and few plasma cells and macrophages. Evidence of an infectious agent was not identified in any image. Scale bars represent 75 μm. Representative images in (A)–(D) are from three moribund animals of the first cohort (n = 25 images). Representative images in (E) and (F) are from two moribund animals of the replication experiment (n = 55 images). For additional images, see also Figure S3. Molecular Therapy 2017 25, 2526-2532DOI: (10.1016/j.ymthe.2017.07.016) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 IL-10 Concentration in CSF and Survival Period in Swine CSF obtained by serial lumbar puncture was measured for IL-10 concentration. Each symbol represents the IL-10 concentration measured from one CSF sample per time point. Abbreviated time series (red) were obtained from animals exhibiting fulminant neurotoxicity, requiring premature euthanasia (time of death, black cross). Gray time series represent animals that survived to final study day 56 (survived, black pig). The first cohort (open symbols) comprised 28 CSF samples from five animals. The second cohort (closed symbols) comprised six CSF samples from two animals. Time is defined as weeks after pIL-10/AAV8 administration. CSF IL-10 concentrations obtained prior to vector administration (baseline) were below the assay detection level (5 × 10−3 pg/mL, horizontal dotted line) for all animals. Molecular Therapy 2017 25, 2526-2532DOI: (10.1016/j.ymthe.2017.07.016) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 Cellular Characteristics of CNS Inflammatory Infiltrates (A–F) Representative images of immunoperoxidase-stained spinal cords depicting CD3+ T lymphocytes (A–C) and CD20+ B lymphocytes (D–F). The majority of infiltrating cells found within the spinal leptomeninges (A and D), perivascular cuffs of spinal gray matter (B and E), and along the central canal (C and F) were T lymphocytes and a minority were B lymphocytes. (A) T lymphocytes infiltrated the leptomeninges diffusely and extended along the Virchow-Robin space. (D) Infiltrating B lymphocytes localized to the CSF-facing side of the leptomeninges facing the i.t. space but did not extend along the Virchow-Robin space. Scale bars represent 150 μm. Molecular Therapy 2017 25, 2526-2532DOI: (10.1016/j.ymthe.2017.07.016) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions