1. Volume 20, Issue 10, Pages 1932-1943 (October 2012)
Thymidine Kinase Suicide Gene-mediated Ganciclovir Ablation of Autologous Gene- modified Rhesus Hematopoiesis 
Cecilia N Barese, Allen E Krouse, Mark E Metzger, Connor A King, Catia Traversari, Frank C Marini, Robert E Donahue, Cynthia E Dunbar 
Molecular Therapy 
Volume 20, Issue 10, Pages (October 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Vectors used for cell and animal studies. (a) MND.TkSR39.LNGFR: The MND backbone vector (5.2 kb) used in the studies contains the myeloproliferative sarcoma virus enhancer (MPSV). The truncated form of the low-affinity nerve growth factor (LNGFR) is the gene marker that is incorporated to the sequence through a viral internal ribosome entry site (IRES) from the encephalomyocarditis virus IRES (600 bp).51 By having an IRES rather than a separate promoter for LNGFR, the expression of LNGFR should be equal than that of the tk gene. The monoclonal antibody CD271 recognizes the tLNGFR on transduced cells. The Tk gene is the SR39 high-affinity mutant of wild-type HSVtk.37 (b) MLV-Tkmut2S-GFP: The viral backbone is based on the MLV virus (7.1 Kb). The Tk gene has a silent T-C transition to remove a cryptic splice donor site. Green fluorescent protein (GFP) as gene marker is driven through the SV40 virus promoter. (c) Amino acid sequence of SR39Tk and Tkmut2S mutants compared to the wild-type HSVtk. The critical sites for GCV interaction are marked as GCV site in gray boxes.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Effect of GCV administration on huCD34+ cells with or without MND.SR39Tk.LNGFR transduction. (a) After transduction and FACS selection for cells expressing surface CD271 (truncated NGFR), huCD34+ cells (closed square) were cultured with different concentrations of GCV for 96 hours, and the % of cell death was determined by flow cytometry via staining for apoptosis markers (Ann-V+ and 7-AAD+). Nontransduced huCD34+ cells (open square) and cells transduced with an MND-GFP vector lacking the HSVtk gene (in gray) were also cultured with GCV for 4 days. Values are mean ± SD (n = 2). *P ≤ (b) After in vitro incubation GCV-transduced (closed square) and nontransduced (open square) huCD34+ were plated in CFU-C assays. Data were collected at two cell concentrations, and assays were plated in triplicate. Values are the mean ± SD. **P ≤ (c) Coculture of mixtures of 10% vector transduced (CD271+) and nontransduced huCD34+ (closed triangle) under escalating GCV doses, compared with transduced (closed square) and nontransduced (open square) huCD34+(n = 1). 7-AAD, 7-amino-actinomycin-D; Ann-V, annexin V; CFU-C, colony-forming units cells; GCV, ganciclovir; GFP, green fluorescent protein; ns, not significant.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Marking levels in macaques receiving cells transduced with the MND.TkSR39.LNGFR vector, before and after GCV treatment. Fate of Tk+PBL in macaques RQ7413 and DC2C before and after GCV treatment. (a) Level of CD271+ PB cells as assessed by flow cytometry is represented before and after the administration of 10 mg/kg/day GCV for 21 days in RQ7413. Dashed black panel represents the GCV infusions. (b) Level of CD271+ PB assessed by flow cytometry in animal DC2C pre and 6 months post-GCV therapy. Two cycles of 15 mg/kg/day GCV for 5 days (solid gray arrow), and 20 mg/kg/day for 5 days (dashed gray arrow) were given 1 month apart. (c) Flow cytometric dot plots of peripheral blood and bone marrow samples stained with the anti-CD271 antibody pre- and post-GCV. Left hand panels: animal RQ7413, right hand panels, animal DC2C. (d) HSVtk copy number as assessed by real time-quantitative PCR (RT-qPCR) analysis for vector sequences before and after GCV therapy. Data shown represents means ± SD of Ct values performed in sample duplicates. Dashed line shows limit of detection 10 copies per 200 ng genomic DNA. GCV, ganciclovir; LNGFR, low-affinity nerve growth factor receptor; PBL, peripheral blood leukocytes; UD, undetectable.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Marking levels in macaques receiving cells transduced with the MLV-Tkmut2S-GFP vector, before and after GCV treatment. (a) Level of GFP+ cells in the peripheral blood of three animals transplanted with MLV-Tkmut2S-GFP-transduced autologous CD34+ cells. Sustained long-term engraftment demonstrated by GFP+ expression in all the animals prior to GCV treatment, and the effect of the GCV treatment (arrows) is shown. Gray arrows represent 10 mg/kg/day GCV administrated intravenous (i.v.) for 21 days at month 10 (macaque RQ7476) or month 8 (macaques 05E119 and RQ7364) after transplant, respectively. Black arrows represent dosing the animals at 15 and 20 mg/kg/day GCV for 5 days, respectively. (b) FACS dot plots illustrating GFP+ PBL before and after GCV therapy. GCV, ganciclovir; GFP, green fluorescent protein; LNGFR, low-affinity nerve growth factor receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Comparison of the killing efficiency of TkSR39 and wild-type Tk proteins in K562 cells. After transduction, cells were exposed to 5 µmol/l GCV in vitro for 96 hours and assayed for cell death markers Ann-V and 7-AAD. *P = Ann-V, annexin V. 7-AAD, 7-amino-actinomycin-D.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Detection of herpes Tk and green fluorescent protein (GFP)-specific interferon-γ (IFN-γ)–producing T cells in transplanted animals. Number of spots in Elispot assays carried out on peripheral blood mononuclear cells in the presence of priming with the specified stimulators. Each bar represents the mean number of spots in duplicate (RQ7413) or triplicate wells ± SD. (a) Macaque RQ7413 transplanted with autologous cells transduced with the MND. TKSR39.LNGFR vector thus cryopreserved peripheral blood mononuclear cell (PBMC) were only primed at increasing concentrations of Tk but not GFP peptides. (b) Macaque RQ7476 transplanted with autologous cells transduced with MLV-Tkmut2S-GFP, primed with both tk and rGFP. (c) Macaque 05E119 transplanted with autologous cells transduced with MLV-Tkmut2S-GFP primed with both Tk and rGFP. (d) Macaque RQ7364 transplanted with autologous cells transduced with MLV-Tkmut2S-GFP, primed with both Tk and rGFP. (e) Detection of antigen-specific IFN-γ–producing T cells in control macaques to polyclonal stimulus (PMA and Con-A), to Tk, and rGFP peptides. Con-A, concanavalin-A, IFN-γ, interferon-γ, PMA, Phorbol 12-Myristate13-Acetate, rGFP, recombinant green fluorescence protein; Tk, thymidine kinase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
T cells subsets in experimental macaques subjected to interferon-γ (IFN-γ) Elispot assay between 4 and 6 months after autologous transplantation with MND-TkSR39-LNGFR (RQ7413), and MLV-Tkmut2S-GFP (RQ7476, 05E119, and RQ7364). (a) Frequency of CD3+ T cells and the CD4+ and CD8+ subpopulations. (b) CD4+/CD8+ ratio of experimental macaques. GFP, green fluorescent protein; LNGFR, low-affinity nerve growth factor receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions