1. Volume 8, Issue 1, Pages 29-41 (July 2003)
Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease 
Michael P Rettig, Julie K Ritchey, Todd E Meyerrose, Jeffrey S Haug, John F DiPersio 
Molecular Therapy 
Volume 8, Issue 1, Pages (July 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Structural features of the ΔCD34-tk retroviral constructs. (A) Sequence differences between the ΔCD34-tk constructs. The ΔCD34-TK fusion protein contains the extracellular, transmembrane, and first 10 aa of the intracellular domain of the human CD34 antigen (ΔCD34), a 24-aa linker (L), and full-length HSV-TK. The aa sequences of the ΔCD34-TK variants are shown below their three-base codons. ΔCD34-tkwt differs from ΔCD34-tk75 by 5 nt substitutions leading to 4 different nonpolar aa in the active site of HSV-tk. ΔCD34-tkwt differs from ΔCD34-tk39SD/SA by 10 nt substitutions leading to 5 different nonpolar aa. The ΔCD34-tk75SD/SA and ΔCD34-tk39SD/SA variants contain third-base degenerate changes (positions 329 and 663) that prevent cryptic splicing of the HSV-tk gene. Bases are numbered from the ATG start codon of wild-type HSV-tk. (B) Schematic representation of retrovirus vectors. In the ΔU3ΔCD34-tk MLV-based vectors, the U3 5′ LTR sequence is deleted and ΔCD34-tk expression is driven from an immediate early CMV enhancer/promoter. In the bicistronic HSV-tk/IRES/GFP and ΔCD34-tk75/IRES/GFP vectors, LTR refers to the MSCV long terminal repeat and IRES to the internal ribosome entry site from the encephalomyocarditis virus. (C) Flow cytometry of ΔCD34-tk75/IRES/GFP-transduced NIH 3T3 cells. NIH 3T3 cells were transduced with the ΔCD34-tk75/IRES/GFP vector and analyzed by flow cytometry 48 h posttransduction using a PE-conjugated anti-CD34 antibody. The percentage of cells in each quadrant is indicated. (D) Flow cytometry of NIH 3T3 cells expressing ΔCD34-tk75t-EGFP. NIH 3T3 cells were stably transfected with the pMX-ΔCD34-tk75t-EGFP vector and analyzed by flow cytometry using a PE-conjugated anti-CD34 antibody. Quadrant gates were set such that>98% of nontransfected 3T3 cells fell in the lower left quadrant.
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
GCV sensitivity of transduced NIH 3T3 cells. (A) FACS analysis of ΔU3ΔCD34-tk-transduced NIH 3T3 cells. NIH 3T3 cells were transduced with ΔU3ΔCD34-tkwt, ΔU3ΔCD34-tk75, ΔU3ΔCD34-tk75SD/SA, or ΔU3ΔCD34-tk39SD/SA retrovirus as indicated. Five days posttransduction, gene-modified cells were isolated by CD34 immunoselection and analyzed by flow cytometry using a PE-conjugated pooled antibody to CD34. Nontransduced cells are shown as a control. (B) FACS analysis of HSV-tk/IRES/GFP-transduced NIH 3T3 cells. Five days posttransduction with HSV-tk/IRES/GFP retrovirus, NIH 3T3 cells were sterile sorted for GFP+ cells using a MoFlo cell sorter. Sorted GFP+ cells (solid line) and nontransduced cells (dashed line) were then analyzed by flow cytometry. (C) GCV sensitivity assay. Purified 3T3 cells expressing wild-type HSV-TK or the ΔCD34-TK variants were evaluated for GCV sensitivity as described under Materials and Methods. The absorbance for each well was recorded and expressed as a percentage of the value for control wells with no GCV added. Each point represents the mean ± SD of eight GCV sensitivity assays, for which each sample was evaluated in triplicate.
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Transduction of primary human T cells. (A) Comparison of activation methods. Human PBMCs were activated with OKT3 (10 ng/ml) or CD3/CD28 beads in the presence of IL-2 (100 U/ml). Two days postactivation, cells were incubated with the ΔU3ΔCD34-tk75 vector for 6 h at an m.o.i. of 1. Cells were then expanded in medium containing IL-2 (100 U/ml) and either OKT3 or CD3/CD28 beads for 5 days and CD34 expression was measured by flow cytometry. The values shown represent means ± SD of four independent experiments. (B and C) Retrovirus titration. CD3/CD28 bead-activated PBMCs were transduced with ΔU3ΔCD34-tk75 (48 h postactivation) at the indicated m.o.i. and CD34 expression was measured 2 (•), 5 (□), or 9 (▴) days posttransduction by flow cytometry. The percentage CD34+ cells is shown in (B) and MFI in (C). (D) Activation kinetics. CD3/CD28 bead-activated PBMCs were transduced with ΔU3ΔCD34-tk75 (m.o.i. = 40) at the indicated times postactivation and CD34 expression was measured 5 days posttransduction by flow cytometry. The percentage CD34+ cells and MFI is shown for each time point. (E and F) Incubation kinetics. CD3/CD28 bead-activated PBMCs were incubated (48 h postactivation) with the ΔU3ΔCD34-tk75 vector (m.o.i. = 20) for the indicated times and CD34 expression was measured 2 (•) or 5 (□) days posttransduction by flow cytometry. The percentage CD34+ cells is shown in (E) and MFI in (F). The data in (B–F) represent the means ± SD of single experiments using two different donors for which each sample was assayed in triplicate.
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
CD34 immunomagnetic selection. (A) Flow cytometry. Representative flow cytometry from a single donor using the optimized purification protocol. Cell staining was performed using a PE-conjugated pooled antibody to CD34 and FITC-conjugated anti-CD8 mAb. (B) Optimization of CD34 immunoselection. Human PBMCs, stimulated 48 h with CD3/CD28 beads, were transduced with the ΔU3ΔCD34-tk75 retrovirus for 4 h at an m.o.i. of 20 and expanded in medium containing IL-2 (100 U/ml) and CD3/CD28 beads for 5 days. Transduced cells (75% CD34+) were then incubated with the indicated amounts of primary anti-CD34 antibody and a secondary antibody coupled to magnetic microbeads. Magnetically labeled cells were isolated on positive-selection columns using a VarioMACS magnetic cell separator (Miltenyi Biotech). The purity of the isolated transduced cells (CD34+ cells) was determined by flow cytometry and the overall yield of the selection procedure from cell counts.
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
GCV sensitivity of transduced primary human T cells. Human PBMCs, stimulated 48 h with CD3/CD28 beads, were transduced with the ΔU3ΔCD34-tkwt, ΔU3ΔCD34-tk75, or ΔU3ΔCD34-tk39SD/SA retrovirus separately, expanded 2–4 days in medium containing IL-2 and CD3/CD28 beads, and then purified by CD34 immunoselection. Selected T cells were then evaluated for GCV sensitivity as described under Materials and Methods. The absorbance for each well was recorded and expressed as a percentage of the value for control wells with no GCV added. Each point represents the mean ± SD of the indicated number of experiments, for which each sample was assayed in triplicate.
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Lymphocyte subset and Vβ repertoire analyses. Human PBMCs, stimulated 48 h with CD3/CD28 beads, were transduced with the ΔU3ΔCD34-tk75 retrovirus, expanded 2 days in medium containing IL-2 and CD3/CD28 beads, and then purified by CD34 immunoselection. (A) Flow cytometric lymphocyte subset and (B) Vβ repertoire analyses were performed on unmanipulated cells (d0: non), mock-transduced cells (d4: non), transduced cells prior to CD34 immunoselection (d4: pre), and ΔCD34-TK75-selected cells (d4: positive). Each time point was assayed in triplicate, and each bar represents the mean ± SD of three different donors.
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Evaluation of ΔCD34-tk75 copy number in primary human T cells. (A) Standard curves for quantitation of albumin and ΔCD34-tk75 by real-time PCR. Serial dilutions of human DNA (for albumin standard curve) or DNA from a clonally expanded NIH 3T3 cell containing a single ΔCD34-tk75 vector integrant were amplified by PCR as described under Materials and Methods. Standard curves for each PCR assay were obtained by linear regression analysis of measured CT values versus the input amount of DNA. Each point represents the mean ± SD of four independent experiments, for which each standard was assayed in triplicate. (B) Relationship between the integrated copy number and ΔU3ΔCD34-tk75 transduction efficiency. Human PBMCs from four different donors were stimulated 48 h with CD3/CD28 beads and transduced with various concentrations of ΔU3ΔCD34-tk75 retrovirus. The percentage of CD34+ T cells was measured 5 days posttransduction by flow cytometry and gene-modified cells were purified to>98% by CD34 immunoselection. Ten nanograms of genomic DNA from each selected sample was analyzed by real-time PCR and the amounts of albumin and HSV-tk in each sample were determined by extrapolation of the CT values using the equation to the respective lines generated by the standard curves in each PCR assay. The vector copy number per CD34+ T cell was calculated by dividing the amount of HSV-tk per sample by twice the amount of albumin per sample. Each point represents the mean ± SD of two independent experiments, for which each sample was assayed in triplicate. Data were fit to a second-order polynomial curve (R2 = 0.936).
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

9. FIG. 8
Activation-dependent expression of ΔCD34-tk75. Human PBMCs, stimulated 48 h with CD3/CD28 beads, were transduced with the ΔU3ΔCD34-tk75 retrovirus, expanded 3 days in medium containing IL-2 (50 U/ml) and CD3/CD28 beads, and then purified to>99% by CD34 immunoselection. Selected cells were then cultured in the presence of CD3/CD28 beads and IL-2 (top) or in IL-2 alone (bottom) and ΔCD34-tk75 expression was monitored at the indicated days postselection by flow cytometry. On day 7 postselection, cells cultured in IL-2 alone were sterile sorted for ΔCD34-TK75− cells using a MoFlo cell sorter and maintained in IL-2 alone or reactivated with CD3/CD28 beads for 4 days as indicated. The percentage CD34+ cells is shown in the upper left and MFI in the upper right of each FACS analysis. Similar results were obtained in two independent experiments.
Molecular Therapy 2003 8, 29-41DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions