Long-term Improvements in Lifespan and Pathology in CNS and PNS After BMT Plus One Intravenous Injection of AAVrh10-GALC in Twitcher Mice  Mohammad A Rafi, Han Zhi Rao, Paola Luzi, David A Wenger  Molecular Therapy  Volume 23, Issue 11, Pages 1681-1690 (November 2015) DOI: 10.1038/mt.2015.145 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Effects of bone marrow transplantation (BMT) and BMT + AAVrh10 treatments on lifespan, weight and gait. (a) Survival of the mice treated with BMT and AAVrh10 alone or in combination is compared to untreated affected and wild-type mice. Vertical blue and green upticks represent mice still living, red upticks refer to mice sacrificed for analysis. The asterisk indicates the mouse that died from gastrointestinal complication. (b) Weights of the mice treated with BMT only (n = 10) and BMT + AAVrh10 (n = 16) are compared to untreated affected (n = 9) and wild-type mice (n = 4). (c) Gait analysis. The footprints of mice were recorded as described in the Materials and Methods section. Footprints are from an 80-day-old wild-type mouse, a 42-day-old untreated affected mouse, a 150-day-old, and a 300-day-old combined treated mouse. Molecular Therapy 2015 23, 1681-1690DOI: (10.1038/mt.2015.145) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Histochemical detection of galactocerebrosidase (GALC) activity using modified X-gal staining. All tissues are from a 150-day-old twi mouse treated with both BMT and AAVrh10. The blue color represents GALC activity. (a) Cortical region of the brain (original magnification ×100); (b) cross section of the cerebellum (original magnification ×40); (c) lumbar region of the spinal cord (original magnification ×100); and (d) cross section of the sciatic nerve (original magnification ×200). Molecular Therapy 2015 23, 1681-1690DOI: (10.1038/mt.2015.145) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Pathological studies in central nervous system (CNS). Tissues from cerebral hemispheres, cerebellum, and spinal cord from different treated mice are compared to affected untreated and wild-type mice. All images are from paraffin sections stained with luxol-fast blue/periodic acid Schiff. Original magnifications for brain and spinal cords samples are ×600 and for cerebellum is ×400. Myelin is stained blue and infiltrating foamy macrophages are stained pink-red. BMT, bone marrow transplantation. Molecular Therapy 2015 23, 1681-1690DOI: (10.1038/mt.2015.145) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Staining of activated microglia/macrophages in CNS of the 150-day-old BMT + AAVrh10-treated mouse compared to the 42-day-old untreated twitcher and 110-day-old wild-type mice. All images are from PFA-fixed frozen sections stained with CD68 antibody. Images (a) through (c) are from cerebral hemisphere white matter (original magnification ×100); images (d) through (f) are from cerebellum (original magnification ×200); images (g) through (i) are from spinal cord white matter (original magnification ×400). As shown in image c and f, brain and cerebellum of the mouse treated with combination of BMT and viral injection, sacrificed at PND150, do not have any CD68-positive cells. However, a section from the lumbar region of the spinal cord does show some staining for CD68 (i). BMT, bone marrow transplantation; CNS, central nervous system; PFA, paraformaldehyde. Molecular Therapy 2015 23, 1681-1690DOI: (10.1038/mt.2015.145) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Astrocyte activation. All images are from PFA-fixed frozen sections stained with glial fibrillary acidic protein antibody that detects astrocytes. Image (a) is from a 110-day-old wild-type mouse, image (b) from 42-day-old untreated twi, image (c) from the 150-day-old BMT + AAVrh10-treated mouse, and image (d) from 224-day-old BMT + AAVrh10-treated mouse (all original magnifications are ×200). As shown in images c and d, the characteristic astrogliosis seen in the untreated twi mouse (image b) appears normal in the combined treated mice. BMT, bone marrow transplantation; PFA, paraformaldehyde. Molecular Therapy 2015 23, 1681-1690DOI: (10.1038/mt.2015.145) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Pathological studies of peripheral nervous system. Cross sections from sciatic nerves of different treated mice are compared to the similar sections from affected untreated and wild-type mice. All images are from paraffin sections stained with luxol-fast blue/periodic acid Schiff. Original magnification for all tissues is ×1,000. The wild-type mouse shows normal myelination (a) while the 42-day-old untreated twi (b) has essentially no myelin and many macrophages. Also the 98-day-old twi mouse treated only with BMT (c) has lost essentially all myelin and is comparable to the untreated affected mouse. In contrast, sciatic nerves from mice of different ages treated with combined BMT/AAVrh10 (d–f) have completely normal looking myelin, and are comparable to the wild-type mouse. BMT, bone marrow transplantation. Molecular Therapy 2015 23, 1681-1690DOI: (10.1038/mt.2015.145) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Ultra-structural studies of the peripheral nervous system. (a–d) Toluidine blue stained semi-thin sections of sciatic nerves from a wild-type mouse (a), untreated twi mouse (b), 150-day-old (c) and 334-day-old combined treated mice (d) are shown. Dark and fuzzy-looking axons in the 334-day-old treated mouse seem to be crush artifacts. Pictures from all sections are taken at ×600 magnification. (e–h) Electron micrographs from the same mice described above. The electron micrograph from a wild-type mouse shows a normal appearing density of large and small myelinated axons with intact axoplasm (e). While the image from untreated twi mouse rarely shows myelin structures and is filled with characteristic inclusions (f), the images from 150- and 334-day-old mice present normal looking myelinated axons with no infiltration of endoneurial macrophages (g,h). All original magnifications are ×4,000. Molecular Therapy 2015 23, 1681-1690DOI: (10.1038/mt.2015.145) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions