Volume 23, Issue 5, Pages 835-844 (May 2015) Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice  Maria Dahl, Alexander Doyle, Karin Olsson, Jan-Eric Månsson, André R A Marques, Mina Mirzaian, Johannes M Aerts, Mats Ehinger, Michael Rothe, Ute Modlich, Axel Schambach, Stefan Karlsson  Molecular Therapy  Volume 23, Issue 5, Pages 835-844 (May 2015) DOI: 10.1038/mt.2015.16 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 A low percentage of normal macrophages rescues the Gaucher disease phenotype. (a) This panel demonstrates the experimental design. The recombination efficiency of the glucosidase β-acid (GBA) gene was determined by DNA polymerase chain reaction (PCR) of clonally derived individual hematopoietic colonies from GBAflox/flox and GBAnull/flox mice at 6 months of age, bulk hematopoietic cells were analyzed for levels of GCase and GluCer. (b) The relative GCase activity in bone marrow (BM), liver, and spleen was determined in WT, GBAflox/flox, and GBAnull/flox mice (n = 3 per group) and (c) substrate accumulation in the tissues (WT, n = 6; GBAflox/flox n = 6; GBAnull/flox, n = 4). (d) BM from GBAflox/flox and GBAnull/flox mice 6 months of age was harvested and colony forming unit-granulocyte macrophage (CFU-GM) assay performed. PCR targeting the excised region of GBA gene in individual colonies (minimum of 25 per group) was performed to determine recombination efficiency (n = 3 per group). *P < 0.05, **P < 0.01, Student t-test. Error bars represent mean + SEM. Molecular Therapy 2015 23, 835-844DOI: (10.1038/mt.2015.16) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Schematic representation of the lentiviral vector design. The figure shows the vectors where the therapeutic vectors contain the codon optimized human glucosidase β-acid (GBA) cDNA driven by the CD68, PGK, and SFFV promoters, respectively. The negative control vector lacks the GBA gene and contains a spacer sequence downstream of the PGK promoter (PGK.Control). cPPT, central polypurine tract; RSV, Rous sarcoma virus enhancer/promoter; SD and SA, splice donor and splice acceptor sites, respectively; Ψ, packaging signal. Molecular Therapy 2015 23, 835-844DOI: (10.1038/mt.2015.16) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Efficient early intervention of Gaucher disease by lentiviral gene transfer. (a) Experimental design of early intervention study. (b) Transduction efficiencies at 5 weeks in peripheral blood (PGK.Control, n = 6; CD68.GBA, n = 6; PGK.GBA, n = 6; SFFV.GBA, n = 5). (c) Percentages of GFP-expressing cells in whole BM at 20 weeks post-transplant (PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 7). (d) Spleen mass: body mass ratio was calculated for the GD1 transplanted mice relative to WT (WT, n = 6; PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 6). (e–g) The GCase activity was determined in BM (e), spleen (f), and liver (g) for the different groups (WT, n = 6; PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 7). (h–j) GluCer accumulation was measured in BM (h), spleen (i), and liver (j) (WT, n = 6; PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 7). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Mann–Whitney U-test. Error bars represent mean + SEM. Molecular Therapy 2015 23, 835-844DOI: (10.1038/mt.2015.16) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Diminished Gaucher cell infiltration in treated animals. Representative histologic analysis of the BM, spleens, and livers from wild-type (WT), GD1 control vector, and therapeutic vector-treated animals 20 weeks posttransplant in the early intervention study. The untreated PGK.Control samples show samples from 6-month-old GD1 mice with infiltration of GluCer- laden cells in bone marrow (BM), spleen, and liver. Periodic acid Schiff staining. Scale bars: upper panel (BM samples); 20 μm, lower panels (spleen and liver samples); 100 μm. Molecular Therapy 2015 23, 835-844DOI: (10.1038/mt.2015.16) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Robust correction of manifest GD1 symptoms by lentiviral gene transfer. (a) Experimental design of late intervention study. (b) Transduction efficiencies at 5 weeks in peripheral blood (PGK.Control, n = 11; CD68.GBA, n = 10; PGK.GBA, n = 6; SFFV.GBA, n = 9). (c) Percentages of GFP-expressing cells in whole BM at 20 weeks post-transplant (PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). (d) Spleen mass: body mass ratio was calculated for the GD1 transplanted mice relative to WT (WT, n = 7; PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). (e–g) The GCase activity was determined in BM (e), spleen (f), and liver (g) for the different groups (WT, n = 7; PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). (h–j) GluCer accumulation was measured in BM (h), spleen (i), and liver (j) (WT, n = 7; PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Mann–Whitney U-test. Error bars represent mean + SEM. Molecular Therapy 2015 23, 835-844DOI: (10.1038/mt.2015.16) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 The CD68 promoter generates relatively high GFP transgene expression in myeloid cells and macrophages. The median fluorescence activity of GFP normalized to PGK.Control in BM macrophages (Mac1+ F4/80+), monocytes (Mac1+), granulocytes (Gr1+), and T-and B-cells (CD3+ CD19+) (PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). Error bars represent mean + SEM. Molecular Therapy 2015 23, 835-844DOI: (10.1038/mt.2015.16) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Gene-corrected Gaucher cells exhibit a typical lentiviral insertion profile. (a) Integration analysis was performed on bone marrow (BM) from 10- to 13-month-old mice in the treatment study at 20 weeks posttransplantation. Five mice per vector group were analyzed. (b) Vector copy number was determined in BM. (c,d) The differences in the polyclonal background (<10 reads) and the amount of prominent insertions (>100 reads). Kruskal–Wallis analysis with Dunn's correction for multiple comparisons. Molecular Therapy 2015 23, 835-844DOI: (10.1038/mt.2015.16) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions