1. Deletions within the HSV-tk transgene in long-lasting circulating gene-modified T cells infused with a hematopoietic graft
by Marina Deschamps, Patricia Mercier-Lethondal, Jean Marie Certoux, Carole Henry, Bruno Lioure, Céline Pagneux, Jean Yves Cahn, Eric Deconinck, Eric Robinet, Pierre Tiberghien, and Christophe Ferrand
Blood
Volume 110(12):
December 1, 2007
©2007 by American Society of Hematology

2. Transgene walking PCR assay.
Transgene walking PCR assay. Starting from the NeoR gene, known to be present and expressed, 9 successive PCRs in sequence (W-01 to W-09) were designed to cover all transgene sequences to the LTR. PCR W-04 enabled detection of the spliced truncated HSV-tk gene. Different lengths enabled rapid determination of the 9 PCR products. In the case of negative PCRs (as illustrated for W-03 to W-04), secondary PCR products, obtained using transgene walking primers flanking the detected deletion area (del-F4 and del-R3 in this example), were cloned and sequenced.
Marina Deschamps et al. Blood 2007;110:
©2007 by American Society of Hematology

3. Monitoring of NeoR+ persistently circulating GMCs
Monitoring of NeoR+ persistently circulating GMCs. (A) Kinetics of persistently circulating NeoR+ GMCs in 4 patients with a follow-up of more than 10 years. △ and ● indicate the absolute number of lymphocytes and GMCs, respectively (right y-axis); □ indicat...
Monitoring of NeoR+ persistently circulating GMCs. (A) Kinetics of persistently circulating NeoR+ GMCs in 4 patients with a follow-up of more than 10 years. △ and ● indicate the absolute number of lymphocytes and GMCs, respectively (right y-axis); □ indicates the percentage of GMCs among circulating lymphocytes (assuming that there is one transgene copy of per cell) (left y-axis). The last analysis date was day 3557 (118.5 months), 3228 (118.5 months), 3379 (112.6 months), and 2063 (68.76 months) for patients 6, 7, 8, and 9, respectively. (B) Absence of T-cell clonality. Clonality was determined by PCR analysis of TCRγ gene rearrangements within donor GMCs before infusion, within PBMCs, and within GMCs G418-reselected at different time points after BMT, using 2 Vγ-Jγ PCR mixes (PCR A-TCRg: V γ 1f + V γ 10 − J γ 1.1/2.1 + J γ 1.3/2.3; PCR B-TCRg: V γ 9 + V γ 11 − J γ 1.1/2.1 + J γ 1.3/2.3). Both PCRs used consensus-specific primers from Vγ families, allowing coverage of most of the TCRγ rearrangements (sensitivity, 10−2 to 10−3). The Gaussian profile confirms the absence of detectable clonal expansion. Multiple peaks without Gaussian distribution were in favor of an oligoclonal T-cell population. Polyclonal and monoclonal controls were DNA extracted from tonsil and Jurkat cell lines, respectively. N/A indicates not analyzable.
Marina Deschamps et al. Blood 2007;110:
©2007 by American Society of Hematology

4. The NeoR transgene is expressed late after transplantation.
The NeoR transgene is expressed late after transplantation. (A) Synopsis of ex vivo GMC G418 reselection and cloning. The process consists of a 7-day polyclonal (CD3-CD28 beads + IL-2) expansion followed by a 5-day selection in the presence of G418 before cloning. A representative agarose gel electrophoresis of qualitative NeoR PCR is shown. When possible, QPCR for the NeoR gene was applied after the selection step to quantify the selection efficiency. After cloning, screening was performed with both qualitative PCR assays to confirm the presence of the NeoR and HSV-tk genes. (B) Representative 2% agarose gel electrophoresis of screening. Except for 1 clone positive for the truncated form of the HSV-tk gene (311 bp of PCR product), all clones were HSV-tk−/NeoR+. Vertical lines have been inserted to indicate a repositioned gel lane.
Marina Deschamps et al. Blood 2007;110:
©2007 by American Society of Hematology

5. Deletions occur within the HSV-tk gene.
Deletions occur within the HSV-tk gene. (A) Agarose gel electrophoresis of PCR performed on DNA of an integral transgene. The 9 PCRs were performed on DNA extracted from 100% G1TKSvNa-transduced HUT cells. As expected, PCR W-04 produced 2 PCR products corresponding to the full-length and truncated HSV-tk gene. (B) Different deletion profiles (WP-01 to WP-04) obtained from clones of different patients. The absence of PCR products in successive PCRs delimited the deletion area. MW indicates molecular weight marker 100-bp DNA ladder. (C) Schematic representation of deletion areas on the G1TKSvNa vector linear map.
Marina Deschamps et al. Blood 2007;110:
©2007 by American Society of Hematology

6. Recombination motif sequences may explain the deletion mechanism.
Recombination motif sequences may explain the deletion mechanism. (A) Sequencing showed that the junction area corresponded to the homologous motif sequence for 3 of 4 patients and that every clone from the same patient carried the same deletion. (B) Size of deletion and precise homologous motif sequence for each patient.
Marina Deschamps et al. Blood 2007;110:
©2007 by American Society of Hematology

7. All the NeoR+/HSV-tk− clones from the same patient descend from the same circulating GMC. All 31 NeoR+/HSV-tk− clones were screened with the 4 retroviral insertion-specific PCRs. PCRs were performed with a LTR-specific reverse primer and a forward primer co...
All the NeoR+/HSV-tk− clones from the same patient descend from the same circulating GMC. All 31 NeoR+/HSV-tk− clones were screened with the 4 retroviral insertion-specific PCRs. PCRs were performed with a LTR-specific reverse primer and a forward primer complementary to the genomic sequence where the retroviral sequence was inserted, previously identified after cloning/sequencing of the LAM PCR product. A PCR product of the expected length was detected in every clone of 1 patient and was absent in the 3 others. Vertical lines have been inserted to indicate a repositioned gel lane.
Marina Deschamps et al. Blood 2007;110:
©2007 by American Society of Hematology

8. Deletions occurred during the ex vivo production of GMCs
Deletions occurred during the ex vivo production of GMCs. Using nested PCR assays with a second run using a specific primer spanning the junction sequence, no deletions within packaging cell line DNA (PC) were detected.
Deletions occurred during the ex vivo production of GMCs. Using nested PCR assays with a second run using a specific primer spanning the junction sequence, no deletions within packaging cell line DNA (PC) were detected. PCR products were detected within GMC products before infusion (D0), as well as 1, 3, 6, and 9 years (Y) after infusion. Specific PCR was applied to GMC samples of the 4 patients, enabling demonstration that deletions were patient specific. ND indicates not done. Vertical lines have been inserted to indicate a repositioned gel lane.
Marina Deschamps et al. Blood 2007;110:
©2007 by American Society of Hematology