Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells by Thasia Leimig, Linda Mann, Maria del Pilar Martin, Erik Bonten, Derek Persons, James Knowles, James A. Allay, John Cunningham, Arthur W. Nienhuis, Richard Smeyne, and Alessandra d'Azzo Blood Volume 99(9):3169-3178 May 1, 2002 ©2002 by American Society of Hematology

Retrovirally transduced PPCA−/− BM cells restore cathepsin A activity in transplanted PPCA−/− recipients.(A) Schematic diagram of the retroviral bicistronic construct encoding the human PPCA cDNA. Retrovirally transduced PPCA−/− BM cells restore cathepsin A activity in transplanted PPCA−/− recipients.(A) Schematic diagram of the retroviral bicistronic construct encoding the human PPCA cDNA. In this vector, expression of both the human PPCA and the GFP marker is driven by the viral LTR. Translation of GFP is initiated at the internal ribosomal entry site (IRES). (B) Platelets, white blood cells (WBCs), and erythrocytes (RBCs), obtained from PPCA−/− mice transplanted with either MSCV-GFP– or MSCV-PPCA-GFP–transduced PPCA−/− BM, at different time points after treatment, were FACS sorted and analyzed for GFP expression; 1 month (1 month, 7 mice total); 3 months (3 months, 6 mice total); LT (long-term, ages 6, 8, 9, and 10 months, 5 mice total). (C) Cathepsin A (CA) activity was assayed in tissue-homogenates of MSCV-PPCA-GFP–treated mice, at different time points after treatment. Wild-type (WT, 4 mice total) and MSCV-GFP–treated PPCA−/− mice (GFP, 4 mice total), ranging in age between 3 and 8 months, were used as positive and negative controls, respectively. The level of CA activity was independent from the age of the wild-type or MSCV-GFP–treated mice. The inset shows the CA activity in brain homogenates ofPPCA−/− treated and untreated mice, as well as controls. For the CA activity in the thymus of 1-month–treated mice, only one tissue sample was collected and measured; 1 month (1 month, 4 mice total); 3 months (3 months, 3 mice total); LT (long-term, ages 6, 8, 9, and 10 months, 5 mice total). We observed considerable variation in the measured CA activity between mice of the same age group, likely due to differences in engraftment of the transplanted BM cells. The presented data are average values with a typical high and low limit of ± 25% to 50%, which is significantly above the CA activity of the GFP controls. The bars represent SDs. Thasia Leimig et al. Blood 2002;99:3169-3178 ©2002 by American Society of Hematology

Histology of systemic organs from BM-transplanted GS mice Histology of systemic organs from BM-transplanted GS mice.Organs from PPCA −/− mice transplanted with total −/−BM transduced with the MSCV-PPCA (BMT-PPCA) retrovirus were isolated at different time points after treatment. Histology of systemic organs from BM-transplanted GS mice.Organs from PPCA −/− mice transplanted with total −/−BM transduced with the MSCV-PPCA (BMT-PPCA) retrovirus were isolated at different time points after treatment. Hematoxylin and eosin-stained tissue sections of the liver (LIV), kidney (KID), and spleen (SP) from a BMT-PPCA–treatedPPCA −/− mouse killed at 9 months after treatment, and from age-matched wild-type andPPCA −/− mice revealed the complete restoration of normal tissue morphology with BM expressing PPCA, compared to the extensive vacuolation present in thePPCA −/− control mouse. Size bar corresponds to 30 μm. Thasia Leimig et al. Blood 2002;99:3169-3178 ©2002 by American Society of Hematology

Immunostaining of tissue sections from BM-transplanted GS mice with α-32 and α-GFP antibodies.Numerous human PPCA-expressing cells were detected by immunostaining with α-32 antibody, monospecific for the human protein (left panels). Immunostaining of tissue sections from BM-transplanted GS mice with α-32 and α-GFP antibodies.Numerous human PPCA-expressing cells were detected by immunostaining with α-32 antibody, monospecific for the human protein (left panels). In the liver of a BMT-treated mouse killed at 9 months after treatment, strong immunostaining was detected in Kupffer cells, as confirmed by staining of adjacent sections with the macrophage-specific anti–Mac-1 antibody. The clear punctate staining of the hepatocytes demonstrated internalization of the corrective enzyme by these cells. In the kidney the presence of the human PPCA was detected in the proximal convoluted tubules and Bowman capsule. Numerous macrophages and splenocytes in the spleen of transplanted mice were positive for the human protein. Staining of the same tissues with α-GFP antibody (right panels) was restricted to cells in locations consistent with their being of hematopoietic origin. Size bar corresponds to 30 μm. Thasia Leimig et al. Blood 2002;99:3169-3178 ©2002 by American Society of Hematology

Histology of 4 regions of the brain from BM-transplanted GS mice Histology of 4 regions of the brain from BM-transplanted GS mice.Hematoxylin and eosin staining of the choroid plexus (CP), the lateral geniculate nucleus (LGN), the amygdala (AMG), and the deep cerebellar nucleus (CbN) isolated from BMT-PPCA–treated GS mou... Histology of 4 regions of the brain from BM-transplanted GS mice.Hematoxylin and eosin staining of the choroid plexus (CP), the lateral geniculate nucleus (LGN), the amygdala (AMG), and the deep cerebellar nucleus (CbN) isolated from BMT-PPCA–treated GS mouse at 9 months after transplantation showed clear reduction of storage in neural cells compared to an age-matched GS animal. The overall brain architecture was clearly improved due to the clearance of storage material in endothelial cells and perivascular and leptomeningeal macrophages. Storage in some of the neurons in the amygdala and cerebellar nucleus was also cleared. Size bar corresponds to 30 μm. Thasia Leimig et al. Blood 2002;99:3169-3178 ©2002 by American Society of Hematology

Immunostaining of 4 brain regions from BM-transplanted GS mice with α-32 and α-GFP antibodies.α-32 immunostaining of brain sections derived from BMT-PPCA–transplanted mice at 9 months after treatment revealed numerous PPCA+ endothelial cells, perivascular m... Immunostaining of 4 brain regions from BM-transplanted GS mice with α-32 and α-GFP antibodies.α-32 immunostaining of brain sections derived from BMT-PPCA–transplanted mice at 9 months after treatment revealed numerous PPCA+ endothelial cells, perivascular macrophages, sparse neurons as well as the cuboidal epithelium of the choroid plexus and its macrophages (indicated by arrowheads). Similar but more restricted immunostaining was detected in sections of the same regions stained with α-GFP antibody. Size bars correspond to 40 μm. Thasia Leimig et al. Blood 2002;99:3169-3178 ©2002 by American Society of Hematology

PEP19 staining of cerebellar sections from BM-transplanted GS mice PEP19 staining of cerebellar sections from BM-transplanted GS mice.Serial sections of the cerebellum from a 9-month-old GS mouse transplanted with MSCV-PPCA–marked BM cells were stained with anti-PEP19 antibody. PEP19 staining of cerebellar sections from BM-transplanted GS mice.Serial sections of the cerebellum from a 9-month-old GS mouse transplanted with MSCV-PPCA–marked BM cells were stained with anti-PEP19 antibody. Note the dramatic loss of Purkinje cells in an age-matched GS mouse and the significant number of these cells that are retained in the treated animal. Size bars correspond to 60 μm for the upper panels and 30 μm for the lower panels. Thasia Leimig et al. Blood 2002;99:3169-3178 ©2002 by American Society of Hematology

Purkinje cell counts in BM-transplanted GS mice Purkinje cell counts in BM-transplanted GS mice.Purkinje cells were counted in recipient mice at 3 and 9 months after treatment and compared to 3- and 9-month-oldPPCA −/− mice and age-matched controls. Purkinje cell counts in BM-transplanted GS mice.Purkinje cells were counted in recipient mice at 3 and 9 months after treatment and compared to 3- and 9-month-oldPPCA −/− mice and age-matched controls. Purkinje cells were counted at 2 levels: (1) in the paravermis at the point where the lateral cerebellar nuclei first become obvious (M, medial), and (2) in the hemisphere at the level of the dorsal cochlear nucleus (H, hemisphere). The values are expressed as percentage of Purkinje cells counted in the control group. Thasia Leimig et al. Blood 2002;99:3169-3178 ©2002 by American Society of Hematology