Volume 19, Issue 7, Pages 1287-1294 (July 2011) Long-term Regulation of Genetically Modified Primary Hematopoietic Cells in Dogs  Kiyoshi Okazuka, Brian C Beard, David W Emery, Kerstin Schwarzwaelder, Michele R Spector, George E Sale, Christof von Kalle, Hans- Peter Kiem, C Anthony Blau  Molecular Therapy  Volume 19, Issue 7, Pages 1287-1294 (July 2011) DOI: 10.1038/mt.2011.8 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Sustained chemical inducer of dimerization (CID) responsiveness in dogs E900 (left) and E958 (right). Percentages of GFP+ red blood cells (RBCs, top), white blood cells (WBCs, middle), and platelets (Plts, bottom) are indicated on the y axis. X axis indicates the number of days post-transplantation. Black boxes and descending dashed lines indicate periods of CID treatment. The first two courses of CID used AP20187 at a dose of 1 mg/kg twice daily intravenously (i.v.) for 30 days. The third course of CID used an abbreviated subcutaneous (s.c.) regimen of AP20187 (5 mg/kg/day s.c. × 5 days). Hatched lines across the x axis indicates a >5-year period of monitoring without CID treatment. In E900, GFP+ cells remained stable at low levels throughout the >5-year period of monitoring without CID. In contrast, GFP+ cells fell to very low levels in E958 during this interval. Administration of CID courses 4 and 5 in E900 (using AP1903 at 5 mg/kg/day s.c. for 5 days) prompted responses that were much more modest albeit qualitatively similar to course 3. †Euthanasia due to the development of an idiopathic cardiomyopathy. Molecular Therapy 2011 19, 1287-1294DOI: (10.1038/mt.2011.8) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Graphical depiction of percentages of green fluorescent protein positive (GFP+) reticulocytes (diamonds) and red blood cells (RBCs) (squares) in E900 in response to chemical inducer of dimerization (CID) courses 4 and 5. Y axis indicates percentage of GFP+ reticulocytes (left) and red blood cells (right)—note difference in scale. x axis indicates days post-transplantation. Black boxes indicate courses of AP1903 (5 mg/kg subcutaneous (s.c.) daily × 5 days). Molecular Therapy 2011 19, 1287-1294DOI: (10.1038/mt.2011.8) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Heterogeneity among chemical inducer of dimerization (CID) responsive hematopoietic cell clones in dogs E958 (top), E900 (bottom), and H247 (bottom right). Y axis depicts the relative abundance of individual clones over time compared to a cellular gene (KIT). X axis indicates the days post-transplantation, with the hatched lines showing a >5-year interval of observation without CID. Black boxes and descending dashed lines indicate courses of CID treatment as described in the legend for Figure 1. **Clones that were detectable in all three lineages (granulocytes, B cells, and T cells); all other clones were only detected in one or two lineages. Three clones that persisted throughout the life of E900 remained detectable in its secondary recipient, H247, and responded to CID. Molecular Therapy 2011 19, 1287-1294DOI: (10.1038/mt.2011.8) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Absence of myelofibrosis in E900 and E958. Photomicrographs of hematoxylin and eosin stained bone marrow biopsies of E900 (left) and E958 (right) at ×100 and ×250 magnification. Bottom right panel shows ×100 magnification of reticulin stain for E958. Molecular Therapy 2011 19, 1287-1294DOI: (10.1038/mt.2011.8) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions