Ex vivo gene therapy using bone marrow-derived cells: combined effects of intracerebral and intravenous transplantation in a mouse model of niemann–pick disease  Hee-Kyung Jin, Edward H Schuchman  Molecular Therapy  Volume 8, Issue 6, Pages 876-885 (December 2003) DOI: 10.1016/j.ymthe.2003.07.008 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 ASM activities in tissues of transplanted ASMKO and control mice. At (A) 16 and (B) 24 weeks postirradiation, ASM activities were measured in several organs. The histograms show comparisons of the mean ASM activities in the transplanted ASMKO animals (open bars) with the activities detected in the same tissues from untreated ASMKO animals (black bars). Cr, cerebrum; Ce, cerebellum; Li, liver; Sp, spleen; Lu, lung; He, heart; Ki, kidney. Data are expressed as means ± SEM (n = 5). Molecular Therapy 2003 8, 876-885DOI: (10.1016/j.ymthe.2003.07.008) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Production of anti-human ASM antibodies in transplanted ASMKO mice. Immunoprecipitation experiments were carried out using pure ASM as the antigen and serum from treated ASMKO, ASMKO control (ASMKO-rad), and normal control (Normal-rad) mice (24 weeks postirradiation). ASM activities were then determined in the immunoprecipitates and supernatants. Data are expressed as means ± SEM (n = 4). Open bars show activities in the immunoprecipitates and black bars the supernatants. Molecular Therapy 2003 8, 876-885DOI: (10.1016/j.ymthe.2003.07.008) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 (A) Survival of ASMKO mice receiving the dual transplants. By 32 weeks postirradiation, all of the ASMKO irradiation control mice (open circles) had died, while seven of nine transplanted ASMKO (closed circles) and all of the normal (triangles) animals survived. The longest surviving transplanted ASMKO animal lived to 48 weeks postirradiation. N = 9 for each group. (B) Rotarod scores for transplanted ASMKO and control mice. Rotarod scores from the same transplanted (closed circles), ASMKO irradiation control (open circles), and normal (triangles) animals were averaged and plotted beginning at 12 weeks postirradiation. Note that all of the ASMKO irradiation control mice died by 32 weeks postirradiation (see A). Molecular Therapy 2003 8, 876-885DOI: (10.1016/j.ymthe.2003.07.008) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Histopathology and sphingomyelin levels in the spleen and cerebellum of transplanted ASMKO and irradiation control mice. (A) Hematoxylin–eosin (top) and lysenin (bottom) staining of spleen sections from representative transplanted ASMKO, ASMKO irradiation control (ASMKO-rad), and normal irradiation control (Normal-rad) mice at 16 weeks postirradiation. (B) Purkinje cell immunohistochemistry at 16 and 24 weeks postirradiation. Purkinje cells were visualized using anti-calbindin antibodies (top). Sphingomyelin staining was performed using lysenin (bottom). Representative images are shown. Original magnification ×40. Molecular Therapy 2003 8, 876-885DOI: (10.1016/j.ymthe.2003.07.008) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 GFAP immunoreactivity in the cerebellum of ASMKO and normal mice at 16 weeks postirradiation. GFAP-positive astrocytes were increased in the ASMKO irradiation control mice (ASMKO-rad) compared to normal irradiation control mice (Normal-rad). GFAP staining was markedly reduced in the transplanted ASMKO mice compared to untreated ASMKO animals and appeared almost normal. Original magnification ×20. Molecular Therapy 2003 8, 876-885DOI: (10.1016/j.ymthe.2003.07.008) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Expression of human ASM RNA in the cerebellum of transplanted ASMKO mice. (A) Antisense or (B) sense human-specific ASM probes and (C) an antisense mouse-specific ASM probe were used to detect ASM RNA in cerebellar sections of transplanted ASMKO, ASMKO irradiation control, and normal irradiation control mice by in situ hybridization. Original magnification ×40. Molecular Therapy 2003 8, 876-885DOI: (10.1016/j.ymthe.2003.07.008) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 7 The number of Purkinje cells expressing human ASM RNA at 24 weeks postirradiation. (A) Schematic depiction of a mouse brain showing the location of the hippocampal (circle) and cerebellar (square) injection sites. Nos. 1–4 indicate the locations of sagittal sections used for the quantification studies shown in C. Positions 2 and 3 are 300 μm from the cerebellar injection site and positions 1 and 4 are 600 μm. (B) Schematic depicting the depth and relative location of the cerebellar injection site, including the individual lobes. (C) Quantification of the Purkinje cells expressing human ASM was performed as described under Materials and Methods. A–E refer to the individual cerebellar lobes. Positions 1–4 (A) are indicated by diamonds, squares, triangles, and circles, respectively. For each lobe and position, at least five sections were analyzed and a total of 100 Purkinje cells were counted. Data are expressed as means ± SEM. Molecular Therapy 2003 8, 876-885DOI: (10.1016/j.ymthe.2003.07.008) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions