Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs by Chiara Dell'Agnola, Zejing Wang, Rainer Storb, Stephen J. Tapscott, Christian S. Kuhr, Stephen D. Hauschka, Richard S. Lee, George E. Sale, Eustacia Zellmer, Serina Gisburne, Janet Bogan, Joe N. Kornegay, Barry J. Cooper, Theodore A. Gooley, and Marie-Térèse Little Blood Volume 104(13):4311-4318 December 15, 2004 ©2004 by American Society of Hematology

DMD genotyping. DMD genotyping. (A) Genomic DNA extracted from blood of wild-type (WT), heterozygote (carrier), and null (affected or xmd) dogs was used for PCR amplification.8 The PCR product containing the polymorphic Sau96I site (B) created by the mutation in the dystrophin gene was digested, electrophoresed, and visualized with ethidium bromide. The genotype of one DLA-matched pair of donor and recipient is shown here. D indicates donor; R, recipient. The wild-type band (310 bp) and the mutant band (150 bp) are marked with arrows. (B) Schematic of the wild-type and xmd mutant alleles. The cxmd allele has a point mutation (a to g) in the consensus splice acceptor site of intron 6. As a consequence, alternative splicing either skips exon 7 entirely (solid line) or splices into a cryptic site 5 nucleotides 3-prime of the original splice site (dashed line). Both of these predominant splice variants introduce frame-shifts and early termination codons. Exon probes (TAMRA and MGB) were designed to hybridize to the wild-type junction between exons 6 and 7 and to distinguish between normally spliced mRNA and the splice variants created by the cxmd mutation. Chiara Dell'Agnola et al. Blood 2004;104:4311-4318 ©2004 by American Society of Hematology

Transplant scheme. Transplant scheme. The xmd dogs were given 920 cGy or 200 cGy TBI on day 0, followed by HCT with G-CSF–mobilized PBMCs and bone marrow from DLA-identical littermates. Following one muscle biopsy, 4 recipients were given G-CSF for 5 days. Myofiber transplant study was used in 3 recipient dogs 9 months after HCT. Muscle biopsies were performed before and after HCT at different time points. CSP was used for postgrafting immunosuppression. TBI indicates total body irradiation; HC transplant source, G-CSF–mobilized PBMCs and bone marrow cells from DLA-identical littermate; MB, muscle biopsy; CSP, cyclosporine, 15 mg/kg orally twice a day, days -2 through +35; G-CSF, granulocyte colony-stimulating factor, 5 mg/kg subcutaneously twice a day for 5 days; and MFE, muscle fiber transplant. Chiara Dell'Agnola et al. Blood 2004;104:4311-4318 ©2004 by American Society of Hematology

Hematopoietic cell recovery after HCT Hematopoietic cell recovery after HCT. Representative example of peripheral blood platelet (○), granulocyte (▪), and lymphocyte (▴) changes in the xmd dogs conditioned with 920 cGy (A) and 200 cGy (B) TBI and given a hematopoietic cell graft from DLA-identi... Hematopoietic cell recovery after HCT. Representative example of peripheral blood platelet (○), granulocyte (▪), and lymphocyte (▴) changes in the xmd dogs conditioned with 920 cGy (A) and 200 cGy (B) TBI and given a hematopoietic cell graft from DLA-identical littermates on day 0. Chiara Dell'Agnola et al. Blood 2004;104:4311-4318 ©2004 by American Society of Hematology

Hematopoietic donor chimerism in 7 dogs. Hematopoietic donor chimerism in 7 dogs. Percent donor peripheral granulocytes (A) and mononuclear cells (B) after HCT measured weekly in 7 xmd dogs. All dogs achieved sustained and stable chimerism after 5 weeks. Chiara Dell'Agnola et al. Blood 2004;104:4311-4318 ©2004 by American Society of Hematology

Dystrophin-positive fibers before and after HCT and at autopsy. Dystrophin-positive fibers before and after HCT and at autopsy. Immunofluorescence staining was performed on muscle tissue sections from pre- and posttransplantation biopsies; necropsy and wild-type dogs were used as positive controls. Sartorius from (A) before HCT and (B) bicep from necropsy; (C) diaphragm from necropsy; (D) sartorius muscle from wild-type dog. Normal mouse immunoglobulin G (IgG; Invitrogen) was used as negative control (not shown). Nonfixed sections were blocked in 3% bovine serum albumin in 1 × Dulbecco phosphate-buffered saline (PBS; Gibco) and incubated for 60 minutes at room temperature with mouse anti–human dystrophin monoclonal antibody (NCL-DYS2, 20 μg/mL; Novocastra Laboratories). Sections were then rinsed 3 times for 15 minutes in PBS and incubated in the dark for 30 minutes at room temperature with rabbit anti–mouse Rhodamine-conjugated secondary antibody (1:100 dilution; Invitrogen). Preparations were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI; Sigma). Sections were then rinsed in PBS (3 × 15 minutes) and mounted in Vectashield (Vector Laboratories). Fields were chosen to show rare positive fibers. Original magnification × 20. Chiara Dell'Agnola et al. Blood 2004;104:4311-4318 ©2004 by American Society of Hematology

Clonal analyses of muscle-derived cells from HC transplant xmd recipients. Clonal analyses of muscle-derived cells from HC transplant xmd recipients. Donor or recipient origin of the muscle-derived cell clones was determined by VNTR analyses. Fibroblast (A) or muscle clones (B) were isolated, harvested, and assayed by VNTR analyses (C). The arrows in panel C refer to the donor-specific bands in the satellite cell clones revealing a mixed genotype in 5 of the clones with donor contributions ranging from 2% to 35% (a = 10%, b ≤ 5%, c ≤ 5%, d ≤ 5%, and e = 20%). Original magnification × 40 (A,B). Chiara Dell'Agnola et al. Blood 2004;104:4311-4318 ©2004 by American Society of Hematology

Dystrophin and fetal myosin staining in muscle fiber transplants in xmd hematopoietic chimeras at 3 weeks and 5 months after transplantation of muscle. Dystrophin and fetal myosin staining in muscle fiber transplants in xmd hematopoietic chimeras at 3 weeks and 5 months after transplantation of muscle. Myofiber transplant from original HC donors to the xmd HC transplant recipients was performed on 3 pairs of dogs. Muscle biopsies were taken at 3 weeks (G123) and 5 months (G141 and G142) after transplantation and costained with dystrophin (red), fetal myosin (green, indicating regeneration), and DAPI (blue, showing nuclear staining). Immunofluorescence staining was performed as described in Figure 5, with the following primary antibodies: rabbit anti–mouse dystrophin (1:400, a kind gift from Jeff Chamberlain, University of Washington, Seattle) and mouse anti–rat fetal myosin (1:20, NCL-MHCd; Novocastra Laboratories). Donkey anti–rabbit Rhodamine-conjugated secondary antibody and rabbit anti–mouse FITC-conjugated secondary antibody (1:100 dilution; Invitrogen) were used for detection. Normal mouse and rabbit isotype (Invitrogen) antibodies were used as negative controls. Focal areas of brightly stained dystrophin-positive fibers were noted in panel A and were also positive for fetal myosin staining. Scattered staining for dystrophin and fetal myosin was observed in panel B after 5 months. However, dystrophin-positive fibers were now negative for fetal myosin. Wild-type muscle from G292 was used as control. Chiara Dell'Agnola et al. Blood 2004;104:4311-4318 ©2004 by American Society of Hematology